2-O sulfatase nucleic acid compositions

ABSTRACT

The invention relates to 2-O sulfatase and uses thereof. In particular, the invention relates to recombinantly produced 2-O sulfatase, functional variants and nucleic acid molecules that encode these molecules. The invention also provides methods of using 2-O sulfatase for a variety of purposes, including degrading and analyzing glycosaminoglycans (GAGs) present in a sample. For instance, 2-O sulfatase may be used for determining the purity, identity, composition and sequence of glycosaminoglycans present in a sample. The invention also relates to methods of inhibiting angiogenesis and cellular proliferation as well as methods for treating cancer, neurodegenerative disease, atherosclerosis and microbial infection using 2-O sulfatase and/or GAG fragments produced by degradation with 2-O sulfatase.

RELATED APPLICATIONS

This application is a divisional application of U.S. patent applicationSer. No. 10/753,761 filed on Jan. 7, 2004, currently pending, and whichclaims the benefit under 35 U.S.C. § 119 of U.S. provisional application60/438,810, filed Jan. 8, 2003. Foreign priority benefits are claimedunder 35 U.S.C. §119(a)-(d) or 35 U.S.C. §365(b) to Japanese applicationnumber 2003-271653, filed Jul. 7, 2003. Each of these applications isincorporated herein by reference in its entirety.

GOVERNMENT SUPPORT

Aspects of the invention may have been made using funding from NationalInstitutes of Health Grants GM 57073 and CA90940. Accordingly, theGovernment may have rights in the invention.

FIELD OF THE INVENTION

The invention relates to 2-O sulfatase, related compositions, andmethods of use thereof.

BACKGROUND OF THE INVENTION

Sulfated glycosaminoglycans such as heparin and the related heparansulfate (HSGAGs) are complex, linear carbohydrates possessingconsiderable chemical heterogeneity (Esko, J. D., and Lindahl, U. (2001)J Clin Invest 108(2), 169-73, Shriver, Z., Liu, D., and Sasisekharan, R.(2002) Trends Cardiovasc Med 12(2), 71-72). Their structural diversityis largely a consequence of the variable number and position of sulfatespresent within a single HSGAG chain. Because of their highly anioniccharacter, these polysaccharides historically have been relegated to anexclusively structural role, namely as a sort of hydration gel andscaffold comprising the extracellular matrix (ECM). Contrary to thislimited perception, however, HSGAGs actually play an important anddynamic function in many critical biological processes ranging fromdevelopment (Perrirnon, N., and Bernfield, M. (2000) Nature 404(6779),725-8) and tissue repair (Simeon, A., Wegrowski, Y., Bontemps, Y., andMaquart, F. X. (2000) J Invest Dermatol 115(6), 962-8) to apoptosis(Ishikawa, Y., and Kitamura, M. (1999) Kidney Int 56(3), 954-63, Kapila,Y. L., Wang, S., Dazin, P., Tafolla, E., and Mass, M. J. (2002) J BiolChem 277(10), 8482-91). These polysaccharides are also central playersin several pathological conditions such as cancer (Selva, E. M., andPerrimon, N. (2001) Adv Cancer Res 83, 67-80, Sasisekharan, R., Shriver,Z., Venkataraman, G., and Narayanasami, U. (2002) Nat Rev Cancer 2(7),521-8), angiogenesis (Folkman, J., and Shing, Y. (1992) Adv Exp Med Biol313, 355-64, Vlodavsky, I., Elkin, M., Pappo, O., Aingorn, H., Atzmon,R., Ishai-Michaeli, R., Aviv, A., Pecker, I., and Friedmann, Y. (2000)Isr Med Assoc J 2 Suppl, 37-45), certain neurodegenerative diseases suchas Alzheimers (Cohlberg, J. A., Li, J., Uversky, V. N., and Fink, A. L.(2002) Biochemistry 41(5), 1502-11), athleroscelerosis (Sehayek, E.,Olivecrona, T., Bengtsson-Olivecrona, G., Vlodavsky, I., Levkovitz, H.,Avner, R., and Eisenberg, S. (1995) Atherosclerosis 114(1), 1-8), andmicrobial infectivity (Liu, J., and Thorp, S. C. (2002) Med Res Rev22(1), 1-25). HSGAGs do so as part of proteoglycans found at the cellsurface and within the ECM where they mediate signaling pathways andcell-cell communication by modulating the bioavailability andtemporal-spatial distribution of growth factors, cytokines, andmorphogens (Tumova, S., Woods, A., and Couchman, J. R. (2000) Int JBiochem Cell Biol 32(3), 269-88) in addition to various receptors andextracellular adhesion molecules (Lyon, M., and Gallagher, J. T. (1998)Matrix Biol 17(7), 485-93). HSGAG structure and function areinextricably related.

A study of the HSGAG structure-function paradigm (Gallagher, J. T.(1997) Biochem Soc Trans 25(4), 1206-9) requires the ability todetermine both the overall composition of biologically relevant HSGAGsas well as ultimately ascertaining their actual linear sequence (finestructure). Therefore the availability of several chemical and enzymaticreagents which are able to cleave HSGAGs in a structure-specific fashionhave proven to be valuable. One example of an important class of GAGdegrading enzymes is the heparin lyases (heparinases) originallyisolated from the gram negative soil bacterium F. heparinum (Ernst, S.,Langer, R., Cooney, C. L., and Sasisekharan, R. (1995) Crit Rev BiochemMol Biol 30(5), 387-444). Each of the three heparinases encoded by thismicroorganism cleave both heparin and heparan sulfate with a substratespecificity that is generally based on the differential sulfationpattern which exists within each GAG chain (Ernst, S., Langer, R.,Cooney, C. L., and Sasisekharan, R. (1995) Crit Rev Biochem Mol Biol30(5), 387-444, Rhomberg, A. J., Ernst, S., Sasisekharan, R., andBiemann, K. (1998) Proc Natl Acad Sci USA 95(8), 4176-81). In fact, F.heparinum uses several additional enzymes in an apparently sequentialmanner to first depolymerize and then subsequently desulfateheparin/heparan sulfate. In addition to heparinase I (Sasisekharan, R.,Bulmer, M., Moremen, K. W., Cooney, C. L., and Langer, R. (1993) ProcNatl Acad Sci USA 90(8), 3660-4), we have recently cloned one of theseenzymes, the Δ 4,5 unsaturated glycuronidase (Myette, J. R., Shriver,Z., Kiziltepe, T., McLean, M. W., Venkataraman, G., and Sasisekharan, R.(2002) Biochemistry 41(23), 7424-7434). This enzyme has beenrecombinantly expressed in E. coli as a highly active enzyme. Because ofits rather unique substrate specificity (Wamick, C. T., and Linker, A.(1972) Biochemistry 11(4), 568-72), this enzyme has already proven to bea useful addition to our PEN-MALDI based carbohydrate sequencingmethodology (Venkataraman, G., Shriver, Z., Raman, R., and Sasisekharan,R. (1999) Science 286(5439), 537-42).

SUMMARY OF THE INVENTION

2-O sulfatase has been cloned from the F. heparinum genome and itssubsequent recombinant expression in E. coli as a soluble, highly activeenzyme has been accomplished. Thus in one aspect the invention providesfor a recombinantly produced 2-O sulfatase.

Recombinant expression may be accomplished in one embodiment with anexpression vector. An expression vector may be a nucleic acid for SEQ IDNO:1, optionally operably linked to a promoter. In another embodimentthe expression vector may be a nucleic acid for SEQ ID NO: 3 or avariant thereof also optionally linked to a promoter. In one embodimentthe recombinantly expressed 2-O sulfatase is produced using a host cellcomprising the expression vector. In another embodiment the expressionvector may comprise any of the isolated nucleic acid molecules providedherein. In some embodiments the protein yields using the recombinantlyexpressed 2-O sulfatases provided herein exceed 100 mg of sulfataseenzyme per liter of induced bacterial cultures. In other embodiments theprotein yield is 110, 115, 120, 125, 130, 150, 175, 200 mg or more perliter of induced bacterial culture. In other aspects methods ofachieving such protein yields are provided comprising recombinantlyexpressing 2-O sulfatase and using at least one chromatographic step.

In another aspect of the invention isolated nucleic acid molecules areprovided. The nucleic acid molecules may be (a) nucleic acid moleculeswhich hybridize under stringent conditions to a nucleic acid moleculehaving a nucleotide sequence set forth as SEQ ID NO: 1 or SEQ ID NO: 3,and which code for a 2-O sulfatase, (b) nucleic acid molecules thatdiffer from the nucleic acid molecules of (a) in codon sequence due todegeneracy of the genetic code, or (c) complements of (a) or (b). In oneembodiment the isolated nucleic acid molecule comprises the nucleotidesequence set forth as SEQ ID NO: 1. In another embodiment the isolatednucleic acid molecule comprises the nucleotide sequence set forth as SEQID NO: 3. In still other embodiments the isolated nucleic acid moleculecodes for SEQ ID NO: 2, and in yet other embodiments the isolatednucleic acid molecule codes for SEQ ID NO: 4.

The isolated nucleic acid molecules of the invention are also intendedto encompass homologs and alleles. In one aspect of the invention, theisolated nucleic acid molecules are at least about 90% identical to thenucleotide sequence set forth as SEQ ID NO: 1 or 3. In otherembodiments, isolated nucleic acid molecules that are at least 91%, 92%,93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 1 or 3 aregiven. In still other embodiments the isolated nucleic acid moleculesare at least 99.5% or 99.9% identical to the nucleotide sequence setforth as SEQ ID NO: 1 or 3.

Therefore, in one aspect of the invention a 2-O sulfatase moleculeproduced by expressing the nucleic acid molecules provided herein isgiven. In some embodiments, as described above, the nucleic acidmolecule is expressed recombinantly. In one embodiment the recombinantexpression is carried out in E. coli.

In another aspect the 2-O sulfatase of the invention is a polypeptidehaving an amino acid sequence of SEQ ID NO: 2, or a functional variantthereof. In yet another aspect the polypeptide has an amino acidsequence of SEQ ID NO: 4, or a functional variant thereof. In stillanother aspect of the invention the 2-O sulfatase is an isolated 2-Osulfatase. In yet another embodiment the isolated 2-O sulfatase issynthetic. In yet another aspect of the invention an isolatedpolypeptide which comprises a 2-O sulfatase is also provided. Theisolated polypeptide in some embodiments comprises a 2-O sulfatasehaving an amino acid sequence set forth as SEQ ID NO: 2. In otherembodiments, the isolated polypeptide comprises a 2-O sulfatase whichhas the amino acid sequence as set forth as SEQ ID NO: 4. In still otherembodiments the isolated polypeptide comprises a 2-O sulfatase which hasthe amino acid sequence as set forth as SEQ ID NO: 2 or 4 or functionalvariants thereof.

In one aspect of the invention, therefore, 2-O sulfatase functionalvariants are provided. In one embodiment the 2-O sulfatase functionalvariants include 2-O sulfatases that contain at least one amino acidsubstitution. In another embodiment the 2-O sulfatase functionalvariants contain 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16,17, 18, 19, 20, 25, 30, 40 or more amino acid substitutions. In some ofthese embodiments the 2-O sulfatase functional variants are 2-Osulfatases that function similarly to the native 2-O sulfatase. In otherembodiments the 2-O sulfatase functional variants are 2-O sulfatasesthat function differently than the native 2-O sulfatase. The differentfunction can be, for instance, altered enzymatic activity or differentsubstrate affinity. For example, as described herein, there are specificactive site amino acids that are positioned to interact with specificconstituents of glycosaminoglycans (e.g., Lys 175, Lys 238 with theplanar carboxyl group of the uronic acid; Lys 107 and possibly Thr 104with the 6-O sulfate of the glucosamine; and Lys 134, Lys 308 with the2-O sulfate). Therefore, 2-O sulfatase functional variants can maintainthese residues or contain amino acid substitutions at these residues tomaintain or alter, respectively, the enzyme's function on a specificsubstrate. In yet other embodiments the amino acid substitutions occuroutside of the active and binding sites as described herein. In stillother embodiments the active and binding sites are targeted forsubstitution. In some of the foregoing embodiments the amino acidsubstitutions occur outside of the catalytic domain given in SEQ ID NO:6. In other embodiments the amino acid substitutions occur within thiscatalytic domain. In still other embodiments the choice of amino acidsubstitutions can be based on the residues that are found to beconserved between the various sulfatase enzymes (e.g., see the sequencealignments provided in FIGS. 3, 9 and 16) (e.g., highly conserved His136, His 191, Asp 42, Asp 63, Asp 295). Amino acid substitutions can beconservative or non-conservative.

In one aspect of the invention the amino acid sequence of the isolatedpolypeptide contains (a) at least one residue selected from Arg 86, Asp42, Asp 159, Asp 295, Cys 82, FGly 82, Gln 43, Gln 237, Glu 106, Gln309, His 136, His 296, Leu 390, Leu 391, Leu 392, Lys 107, Lys 134, Lys175, Lys 238, Lys 308 or Thr 104 and (b) at least one amino acidsubstitution. In one embodiment of the invention the amino acid sequenceof the isolated polypeptide contains a Cys 82 residue and at least oneamino acid substitution. In another embodiment the isolated polypeptidecontains a Cys 82 residue which is subsequently modified to formylglycine and at least one amino acid substitution. In still otherembodiments the isolated polypeptide contains a FGly 82 residue and atleast one amino acid substitution.

In another aspect of the invention functional variants include a 2-Osulfatase which contains at least one amino acid residue that has beensubstituted with a different amino acid than in native 2-O sulfatase andwherein the residue that has been substituted is selected from Arg 86,Asp 42, Asp 159, Asp 295, Gln 43, Gln 237, Glu 106, Gln 309, His 136,His 296, Leu 390, Leu 391, Leu 392, Lys 107, Lys 134, Lys 175, Lys 238,Lys 308 and Thr 104.

In another aspect, the invention is a composition comprising, anisolated 2-O sulfatase having a higher specific activity than native 2-Osulfatase. In some embodiments, the 2-O sulfatase has a specificactivity that is at least about 5-fold higher than native 2-O sulfatase.The specific activity of the 2-O sulfatase in other embodiments may be6-, 7-, 8-, 9-, 10-, 11-, 12-, 13-, 14-, 15-, 16-, 17-, 18-, or 19-foldhigher than the specific activity of the native enzyme. In otherembodiments the specific activity may be about 20-, 25-, 30-, 40- or50-fold higher. In one embodiment the 2-O sulfatase has a specificactivity that is about ten fold higher than the specific activity of thenative enzyme.

In another aspect the invention also provides a method of degrading aglycosaminoglycan. The method may be performed by contacting theglycosaminoglycan with a 2-O sulfatase of the invention in an effectiveamount to degrade-the glycosaminoglycan. In other embodiments the methodmay be performed by contacting the glycosaminoglycan with at least oneother glycosaminoglycan degrading enzyme. In some embodiments the atleast one other glycosaminoglycan degrading enzyme is heparinase orglycuronidase. In other embodiments the glycosaminoglycan is contactedwith the at least one other glycosaminoglycan degrading enzymeconcomitantly with the 2-O sulfatase. In still other embodiments theglycosaminoglycan is contacted with the at least one otherglycosaminoglycan degrading enzyme prior to or subsequent to contactingthe glycosaminoglycan with 2-O sulfatase. In still another embodimentthe glycosaminoglycan is contacted with a heparinase prior to contactwith a 2-O sulfatase.

In some embodiments the glycosaminoglycan is a long chain saccharide. Insuch embodiments the glycosaminoglycan is a tetrasaccharide or adecasaccharide. In other embodiments the glycosaminoglycan contains a2-O sulfated uronic acid at the non-reducing end. In still otherembodiments the glycosaminoglycan contains a β1→4 linkage. In yetanother embodiment the glycosaminoglycan is a chondroitin sulfate. Inother embodiments the glycosaminoglycan is a highly sulfatedglycosaminoglycan. In such embodiments the highly sulfatedglycosaminoglycan contains a 6-O sulfated glucosamine. In yet otherembodiments the highly sulfated glycosaminoglycan contains a glucosaminesulfated at the N-position.

In some aspects of the invention degraded glycosaminoglycans prepared bythe methods described herein are provided. In still other aspects of theinvention a composition which contains a degraded glycosaminoglycan isgiven. In still another aspect of the invention the composition is apharmaceutical preparation which also contains a pharmaceuticallyacceptable carrier.

The present invention also provides methods for the analysis of aglycosaminoglycan or group of glycosaminoglycans. In one aspect theinvention is a method of analyzing a glycosaminoglycan by contacting aglycosaminoglycan with the 2-O sulfatase of the invention in aneffective amount to analyze the glycosaminoglycan.

The present invention also provides 2-O sulfatase immobilized on a solidsupport. In another embodiment at least one other glycosaminoglycandegrading enzyme is also immobilized on the solid support.

In one aspect of the invention a method for identifying the presence ofa particular glycosaminoglycan in a sample is provided. In anotheraspect of the invention a method for determining the identity of aglycosaminoglycan in a sample is provided. In yet another aspect of theinvention a method for determining the purity of a glycosaminoglycan ina sample is also provided. In still a further aspect of the invention amethod for determining the composition of a glycosaminoglycan in asample is provided. Yet another aspect of the invention is a method fordetermining the sequence of saccharide units in a glycosaminoglycan. Insome embodiments, these methods can further comprise an additionalanalytical technique such as mass spectrometry, gel electrophoresis,capillary electrophoresis or HPLC.

In another aspect the invention is a method of inhibiting angiogenesisby administering to a subject in need thereof an effective amount of anyof the pharmaceutical preparations described herein for inhibitingangiogenesis.

In another aspect a method of treating cancer by administering to asubject in need thereof an effective amount of any of the pharmaceuticalpreparations described herein for treating cancer is also provided.

Yet another aspect of the invention is a method of inhibiting cellularproliferation by administering to a subject in need thereof an effectiveamount of any of the pharmaceutical preparations described herein forinhibiting cellular proliferation.

In yet another aspect of the invention a method of treatingneurodegenerative disease by administering to a subject in need thereofan effective amount of any of the pharmaceutical preparations describedherein for treating neurodegenerative disease is provided. In oneembodiment the neurodegenerative disease is Alzheimer's disease.

Another aspect of the invention is a method of treating atherosclerosisby administering to a subject in need thereof an effective amount of anyof the pharmaceutical preparations described herein for treatingatherosclerosis.

In another aspect of the invention a method of treating or preventingmicrobial infection by administering to a subject in need thereof aneffective amount of any of the pharmaceutical preparations describedherein for treating or preventing microbial infection is given.

In yet another aspect of the invention a method of controlling apoptosisby administering to a subject in need thereof an effective amount of anyof the pharmaceutical preparations described herein for controllingapoptosis is provided.

In other aspects of the invention methods of repairing tissue orcontrolling development are also provided.

In some embodiments of the methods of the invention the 2-O sulfatase isused concurrently with, prior to or following treatment with at leastone other glycosaminoglycan degrading enzyme. In some embodiments the atleast one other glycosaminoglycan degrading enzyme is heparinase orglycuronidase. In some embodiments of the compositions or pharmaceticalpreparations of the invention other enzymes such as heparinase and/orglycuronidase may be included.

In other aspects of the invention, compositions, pharmaceuticalpreparations and therapeutic methods are provided with/using the 2-Osulfatase or the degraded glycosaminoglycans alone or in combination.

Compositions of any of the 2-O sulfatases, degraded glycosaminoglycans,nucleic acids, polypeptides, host cells or vectors described herein arealso encompassed in the invention. Pharmaceutical preparations of anycomposition provided herein are also provided in some embodiments. Inthese embodiments the pharmaceutical preparations contain apharmaceutically acceptable carrier.

In still another aspect of the invention, a substantially pure,non-recombinantly produced 2-O sulfatase that has a purity that is about3000-fold greater than crude bacterial lysate is provided. In someembodiments the purity of the substantially pure, non-recombinantlyproduced 2-O sulfatase is about 4000-, 5000-, 6000-, 7000-, 8000-, 9000-or 10,000-fold more pure than crude bacterial lysate. In someembodiments the substantially pure, non-recombinantly produced 2-Osulfatase is obtained by a multi-step fractionation method. In oneembodiment the method is a five-step fractionation method. In thisaspect of the invention, the term “substantially pure” means that theproteins are essentially free of other substances to an extent practicaland appropriate for their intended use.

Each of the limitations of the invention can encompass variousembodiments of the invention. It is, therefore, anticipated that each ofthe limitations of the invention involving any one element orcombinations of elements can be included in each aspect of theinvention.

These and other aspects of the invention, as well as various advantagesand utilities, will be more apparent with reference to the detaileddescription of the preferred embodiments.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 provides the results of Flavobacterium 2-O sulfatase purificationand proteolysis. Panel (A) provides the final RP-HPLC chromatography ofblue-Sepharose CL-6B purified sulfatase. Panel (B) illustrates the C4RP-HPLC chromatographic resolution of sulfatase peptides generated by alimit trypsin digestion of the major peak shown in Panel (A).

FIG. 2 provides the F. heparinum 2-O sulfatase coding sequence (openreading frame from genomic clone S4A. The nucleic acid and amino acidsequence (SEQ ID NOs: 1 and 2, respectively) of the full length gene forthe 2-O sulfatase begins with the first methionine (the nucleic acid andamino acid sequences including the sequence upstream of the firstmethionine are provided as SEQ ID NOs: 38 and 39, respectively). Thenucleic acid and amino acid sequence of the truncated 2-O sulfatasewhich lacks the first 24 amino acids (herein referred to as 2-O ΔN¹⁻²⁴)of the full length gene are given as SEQ ID NOs: 3 and 4, respectively.Translation initiation and termination codons are shown in bold. Primersused in original PCR screen are noted by horizontal arrows. Internal NdeI site is double underscored. Corresponding amino acid sequence ofselect sulfatase peptides are boxed. Sulfatase consensus sequenceCXPXRXXXXS/TG (SEQ ID NO: 5) is boxed and shaded with active sitecysteine at position 82 noted by an asterisk. Putative signal sequenceis overscored with predicted peptidase cleavage site represented by avertical arrow.

FIG. 3 depicts a 2-O sulfatase multiple sequence alignment (SEQ ID NOs:40-58). The flavobacterial enzyme is a member of a large sulfatasefamily. Alignment shown excludes 2-O sulfatase carboxy terminus (aminoacids 374-468). The putative active site is boxed with criticallymodified cysteine noted by an asterisk. Invariant residues are shaded indark gray, partial identity in light gray, conservative substitutions incharcoal. Multiple sequence alignment was generated by ClustalW usingonly select bacterial sequences identified from a BLASTP search of theprotein database. Mammalian sulfatases are not included. Most sequenceslisted correspond to the open reading frame of genes to which only aputative sulfatase function has been ascribed. GenBank accession numbersare as follows: AA605721 (Pseuodmonas aeruginoasa.); AL355753(Streptomyces coelicolor); BAB79937 (E. coliO157:H7); AAF72520(Prevotella sp. MdsA gene); AAL:45441 (Agrobacterium tumefaciens);AAL19003 (Salmonella typhimurium).

FIG. 4 provides the results from the purification of recombinant 2-Osulfatase from E. coli lysates by Ni⁺² chelation chromatography. Enzymepurity following each fractionation step was assessed by silver-stainingof 12% SDS-polyacrylamide gels. Approximately 200 ng of total proteinwas loaded in each well. Lane 1, bacterial lysate from uninduced (minusIPTG) control; lane 2, whole cell lysate; lane 3, 20,000×g supernatant(column pre-load); lane 4, eluate from Ni⁺² chelation chromatography;lane 5, 2-O sulfatase following thrombin cleavage to remove NH₂ 6×histidine purification tag. Molecular weight markers (M_(r)) and theircorresponding masses are also shown.

FIG. 5 illustrates the exclusive desulfation of the 2-OH position by therecombinant sulfatase. Panel (A) depicts the enzyme desulfating activityassayed by capillary electrophoresis using the 2-O containingtrisulfated heparin disaccharide ΔU₂₅H_(NS,6S). Panel (B) depicts theactivity using its disulfated counterpart to ΔU_(2S)H_(NS,6S) lacking asulfate at the 2-OH position. Only in Panel (A) is a loss of sulfateobserved. Minus enzyme control is shown as a dotted line.

FIG. 6 provides the in vitro biochemical reaction conditions for therecombinant 2-O sulfatase. Panel (A) illustrates the effect of pH.Sulfatase catalytic efficiency (k_(cat)/K_(m)) was measured as afunction of varying pH from 5 to 8 using two overlapping buffers: 50 mMMES (solid circles) and 50 mM MOPS (open circles). Inset: Relativeeffect of three different assay buffers (each at pH 6.5) on optimalenzyme activity. 1. 50 mM MES; 2. 50 mM imidazole; 3. 50 mM sodiumphosphate. Panel (B) illustrates the effect of ionic strength. Shownhere is % activity normalized to 50 mM NaCi. Panel (C) illustrates theeffect of reaction temperature. Data is normalized to 30° C. activity(100%). The unsaturated disaccharide ΔU_(2S)H_(NS) was used in all threeexperiments.

FIG. 7 illustrates the substrate-product relationship between the 2-Osulfatase and the Δ 4,5 glycuronidase. 2 mM of the unsaturated, 2-Osulfated heparin disaccharide ΔU_(2S)H_(NS) was preincubated with either250 nM Δ 4,5 glycuronidase or 25 nM 2-O ΔN¹⁻²⁴ for two minutes at 30° C.in a 100 μL reaction. Following this preincubation, the reciprocalenzyme was added to the reaction for up to six extra minutes. Δ 4,5glycuronidase activity was measured in real time as the rate ofsubstrate disappearance monitored by the loss of UV absorption at 232nm. Zero time on the x-axis represents the time following thepreincubation during which the second enzyme was added.

FIG. 8 illustrates the results of the tandem use of 2-O sulfatase and Δ4,5 glycuronidase in HSGAG compositional analyses. Panel (A) providesthe results of exhaustively cleaving 200 μg heparin with heparinase I,II and III. These heparinase-generated saccharides were then subjectedto hydrolysis by the Δ 4,5 glycuronidase. Panel (B) provides the resultsof subsequent hydrolysis by 2-O sulfatase after the heparinase treament.Panel (C) illustrates subsequent hydrolysis by 2-O sulfatase and by Δ4,5 glycuronidase added simultaneously. Panel (D) depicts the 7disaccharide peaks (and one tetrasaccharide peak) resolved by capillaryelectrophoresis (each numbered separately). Their compositionalassignments are as follows: ΔU_(2S)H_(NS,6S) (1);ΔUH_(NAc,6S)GH_(NS,3S,6S) tetrasaccharide (2); ΔU_(2S)H_(NS) (3);ΔUH_(NS,6S) (4); ΔU_(2S)H_(NAc,6S) (5); ΔUH_(NS) (6); ΔU_(2S)H_(NAc)(7); and ΔUH_(NAc,6S) (8).

FIG. 9 illustrates the multiple sequence alignment of sulfatases usingClustaiW (SEQ ID NOs:59-62). The sequence of F. heparinum 2-O sulfatase(F2OS) was aligned with human arylsulfatase B (ARSB), humanarylsulfatase A (ARSA) and P. aeruginosa arylsulfatase (PARS). The aminoand carboxyl termini are not shown. The sequence numbers for eachsulfatase are listed on the right. The numbers listed above thealignment correspond specifically to F2OS sequence positions (see FIG. 2above). The critical active site cysteines are highlighted in black.Other highly conserved amino acids are highlighted in gray.

FIG. 10 provides the structural model of 2-O sulfatase and topology ofthe active site. Panel (A) is the ribbon diagram of the proposed 2-Osulfatase structure constructed using homology modeling of the crystalstructure of human arylsulfatase B. The β strands are shown as thickerareas of the ribbon diagram, and the α helices are shown ascylindrically shaped areas. The geminal diol form of the modifiedcysteine is also depicted (rendered as CPK; carbon and oxygen moleculesare shown). The direction of substrate diffusing into the active site isindicated by an arrow. Panel (B) provides the CPK rendering of the topview of the structure shown in Panel (A). The modified cysteine, thesurrounding basic amino acids (Arg, His and Lys), acidic amino acids(Asp, Glu), and Gln and Asn are all shown. Note that the active sitegeminal diol is located in the bottom of a deep cleft.

FIG. 11 depicts the active site amino acids and their interaction withΔU_(2S)H_(NS,6S). Panel (A) is the stereo view of the 2-O sulfataseactive site highlighting important amino acids (shown here by a stickrepresentation). Acidic amino acids (Asp), Gln, Thr, Leu, and FGly 82are depicted. The docked disaccharide is also shown using a stickrepresentation. The sulfur atom of the 2-O sulfate group (next to thelowest positioned oxygen) and oxygen atoms (circled) of the 2-O sulfategroup and the planar carboxyl group are also depicted. Panel (B)provides the schematic representation of the amino acids shown in Panel(A). Potential metal ion coordination is also shown with the divalentcation (Mg²⁺) depicted as a gray circle.

FIG. 12 illustrates the exolytic activity of the 2-O sulfatase byanalyzing the ability of the sulfatase to hydrolyze internallypositioned 2-O sulfates within the AT10 decasaccharide and subsequentcompositional analyses of the heparinase-treated product. Panel (A)shows the AT-10 decasaccharide sequence with PEN-MALDI nomenclature andoutline of experimental design. Panel (B) provides the capillaryelectrophoretogram for both the control and sulfatase pre-treatedsamples along with their saccharide compositional assignments.Heparinase cleavage products following sulfatase pre-treatment are shownas a dashed line (with gray fill). Minus sulfatase control is shown as awhite line (no fill). The pentasulfated tetrasaccharide (4, -7) is alsonoted. Disappearance of the trisulfated disaccharide (D) by one-thirdand the corresponding appearance of the 2-O desulfated product(ΔUH_(NS,6S)) are depicted by vertical arrows. The minor tetrasaccharidecontaminant is noted by an asterisk.

FIG. 13 illustrates the steady-state kinetics for various unsaturateddisaccharide substrates. Panel (A) provides the initial rates determinedusing 25 nM enzyme under standard conditions. Substrate saturation datawere fit to pseudo-first order Michaelis-Menten assumptions using anon-linear least squares analysis. ΔU_(2S)H_(Nac) (A); ΔU_(2S)H_(Nac,6S)(•); ΔU_(2S)H_(NS) (▴); ΔU_(2S)H_(NS,6S) (◯); ΔU_(2S)Gal_(NAc,6S) (+).

FIG. 14 provides the comparable CD spectroscopy of the wild-type 2-OΔN¹⁻²⁴ sulfatase and C82A site-directed mutant—wild-type enzyme (•),C82A mutant (◯). Band intensities are expressed as molar ellipticitieswith units indicated.

FIG. 15 illustrates the identification of 2-O sulfatase active sitemodification (FGly) by chemical labeling and mass spectrometry.Wild-type sulfatase (2-O Δ¹⁻²⁴) and C82A mutant were reacted with TexasRed Hydrazide and subjected to trypsin proteolysis as described inMaterials and Methods. The molecular masses of the resultant peptideswere subsequently characterized by MALDI-MS. Panel (A) shows theunlabeled wild-type sulfatase control. Panel (B) shows the covalentlylabeled wild-type sulfatase. Panel (C) shows the C82A mutant refractoryto chemical labeling. A unique molecular mass signature in Panel (B) isnoted by an asterisk.

FIG. 16 shows a multiple sequence alignment of the sulfatases usingClustalW (SEQ ID NOs: 2 and 63-83). The putative active site is boxed,with critically modified cysteine noted by an asterisk. Invariantresidues are shaded in dark gray, those with partial identity in lightgray, and conservative substitutions in charcoal. Multiple sequencealignment was generated by ClustalW using only select sequencesidentified from a BLASTP search of the protein data base. Mammaliansulfatases are included. Enzymes are abbreviated as follows. FH2S, F.heparinum 2-O-sulfatase; PARS, P. aeruginosa arylsulfatase; MDSA,Prevotella sp. MdsA gene; HGal6S, human N-acetylgalactosamine-6-sulfatesulfatase (chondroitin 6-sulfatase); HARSA, human cerebroside-3-sulfatesulfatase (arylsulfatase A); HARSB, human N-acetylgalactosamine-4sulfate sulfatase (arylsulfatase B); H125, human iduronate-2-sulfatesulfatase; cons, consensus sequence. The GenBank™ protein accessionnumbers for sulfatases listed are as follows: CAA88421, P. aeruginosaarylsulfatase; AAF72520, Prevotella sp. MdsA mucin desulfating gene;AAC51350, Homo sapiens N-acetylgalactosamine-6-sulfate sulfatase;AAB03341, H. sapiens cerebroside-3-sulfate sulfatase (arylsulfatase A);AAA51784, H. sapiens N-acetylgalactosamine-4-sulfate sulfatase(arylsulfatase B); AAA63197, H. sapiens iduronate-2-sulfate sulfatase.

DETAILED DESCRIPTION OF THE INVENTION

Heparin and heparin sulfate glycosaminoglycans (HSGAGs) are structurallycomplex linear polysaccharides (Esko, J. D., and Lindahl, U. (2001) JClin Invest 108(2), 169-73, Lindahl, U., Kusche-Gullberg, M., andKjellen, L. (1998) J Biol Chem 273(39), 24979-82) comprised of repeatingdisaccharides of uronic acid (α-L-iduronic or β-D-glucuronic) linked 1→4to α-D-glucosamine. The extensive chemical heterogeneity of thesebiopolymers derives from both the variable number of their constituentdisaccharides as well as the combinatorial potential for chemicalmodification at specific positions within each of these building blocks.Such modifications include acetylation or sulfation at the N-position ofthe glucosamine, epimerization of glucuronic acid to iduronic acid andadditional sulfations at the 2-O position of the uronic acid in additionto the 3-O, 6-O position of the adjoining glucosamine. It is a highlyvariable sulfation pattern, in particular, that ascribes to each GAGchain a unique structural signature. In turn, this signature dictatesspecific GAG-protein interactions underlying critical biologicalprocesses related to cell and tissue function.

One of the more formidable challenges currently facing the glycobiologyfield is the design of effective analytical methods to study thisstructure-function relationship at the molecular level. Given thiscritical structure-function relationship of GAG sulfation, enzymes whichcan hydrolyze these sulfates in a structurally-specific manner becomeimportant in several ways. To begin with, the systematic desulfation ofGAGs at discrete positions is central to GAG catabolism that occurs indivergent organisms ranging from bacteria to mammals. In addition, thein vivo desulfation of intact GAG chains both at discrete chemicalpositions and in a cell specific, temporally relevant context is alsolikely to serve as an important molecular switch for abrogating targetedGAG-protein interactions.

2-O sulfatase is a desulfating enzyme that can be now added to therepertoire of enzymes used to analyze GAGs and degrade them in aspecific manner. As used herein, the term “degraded glycosaminoglycan”or “GAG fragment” is intended to encompass a glycosaminoglycan that hasbeen altered from its original form by the activity of a 2-O sulfataseor other enzyme that can act thereon. The degraded glycosaminoglycanincludes glycosaminoglycans that have been altered by the activity of a2-O sulfatase in some combination with other glycosaminoglycan degradingenzymes as described herein. The degraded glycosaminoglycan may bedesulfated, cleaved or desulfated and cleaved. Any of the degradedproducts produced by the activity of the 2-O sulfatase and/or otherenzymes on the glycosaminoglycan are intended to be used in thecompositions, pharmaceutical preparations and methods of the invention.In addition, this sulfatase can be used in treatment methods along withthe GAG fragments they degrade. 2-O sulfatase is a member of a largeenzyme family that hydrolyze a wide array of sulfate esters (for areview, see (Parenti, G., Meroni, G., and Ballabio, A. (1997) Curr OpinGenet Dev 7(3), 386-91, von Figura, K., Schmidt, B., Selmer, T., andDierks, T. (1998) Bioessays 20(6), 505-10)). This enzyme exhibits 2-Ospecific sulfatase activity as measured using the trisulfated,unsaturated heparin disaccharide ΔU_(2S)H_(NS,6S) as a substrate(described below). The activity of the enzyme is not limited to 2-Odesulfation-alone, however, as 2-O sulfatase was found to hydrolyze atthe 6-O and 2N positions of glucosamine. 2-O sulfatase can be used tohydrolyze heparin and chondroitin disaccharides and can also desulfateGAGs with longer chain lengths such as tetra- and decasaccharides.Furthermore, 2-O sulfatase has been found to work with other GAGdegrading enzymes such as heparinases and Δ 4,5 glycuronidase and can beused in conjunction with these other enzymes as described herein.

Like the Δ 4,5 glycuronidase, which we have recently cloned andexpressed (Myette, J. R., Shriver, Z., Kiziltepe, T., McLean, M. W.,Venkataraman, G., and Sasisekharan, R. (2002) Biochemistry 41(23),7424-7434), we have successfully cloned from Flavobacterium heparinumand expressed the 2-O sulfatase in E. coli, from which milligramquantities of highly active, soluble enzyme were readily purified. Aswas also the case for the glycuronidase, we found that the yield ofsoluble recombinant enzyme was greatly improved by the engineeredremoval of the hydrophobic N-terminal signal sequence comprised of thefirst 24 amino acids. This signal sequence was predicted by the vonHeinje method which also identified the likely signal peptidase cleavagerecognition sequence AXAXA. By engineering a 2-O sulfatase N-terminaltruncation lacking this sequence (herein referred to as 2-O ΔN¹⁻²⁴), weachieved protein yields exceeding 100 mg of relatively pure sulfataseper liter of induced bacterial cultures using a single chromatographicstep.

The invention, therefore, provides, in part, a recombinantly produced2-O sulfatase. As used herein, a “recombinant 2-O sulfatase” is a 2-Osulfatase that has been produced through human manipulation of thenucleic acid that encodes the enzyme. The human manipulation usuallyinvolves joining the nucleic acid that encodes the 2-O sulfatase to thegenetic material of a different organism and, generally, a differentspecies. “Recombinant” is a term of art that is readily known to one ofskill, and techniques for the recombinant expression of 2-O sulfataseare readily available to those of skill in the art and include thosedescribed in Sambrook et al., Molecular Cloning—A Laboratory Manual,Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y., (1989) orCurrent Protocols in Molecular Biology Volumes 1-3, John Wiley & Sons,Inc. (1994-1998). Other techniques for recombinant expression includingexamples of expression systems are described further below.

As provided herein, recombinant technology can be used to produce a 2-Osulfatase encoded by the nucleic acid sequence of SEQ ID NO: 1 or havingthe amino acid sequence of SEQ ID NO: 2. In other aspects of theinvention a 2-O sulfatase encoded by the nucleic acid sequence of SEQ IDNO: 3 or having the amino acid sequence of SEQ ID NO: 4 can be prepared.The 2-O sulfatase as provided herein is, in general, produced throughthe manipulation of isolated nucleic acids.

The invention also provides the isolated nucleic acid molecules thatcode for a 2-O sulfatase as described herein. The term “isolated nucleicacid”, as used herein, means: (i) amplified in vitro by, for example,polymerase chain reaction (PCR); (ii) recombinantly produced by cloning;(iii) purified, as by cleavage and gel separation; or (iv) synthesizedby, for example, chemical synthesis. An isolated nucleic acid is onewhich is readily manipulable by recombinant DNA techniques well known inthe art. Thus, a nucleotide sequence contained in a vector in which 5′and 3′ restriction sites are known or for which polymerase chainreaction (PCR) primer sequences have been disclosed is consideredisolated but a nucleic acid sequence existing in its native state in itsnatural host is not. An isolated nucleic acid may be substantiallypurified, but need not be. For example, a nucleic acid that is isolatedwithin a cloning or expression vector is not pure in that it maycomprise only a tiny percentage of the material in the cell in which itresides. Such a nucleic acid is isolated, however, as the term is usedherein because it is readily manipulable by standard techniques known tothose of ordinary skill in the art.

According to the invention, isolated nucleic acid molecules that codefor a 2-O sulfatase include: (a) nucleic acid molecules which hybridizeunder stringent conditions to a molecule selected from a groupconsisting of the nucleotide sequences set forth as SEQ ID NO: 1 and 3and which code for a 2-O sulfatase or parts thereof, (b) deletions,additions and substitutions of (a) which code for a 2-O sulfatase orparts thereof, (c) nucleic acid molecules that differ from the nucleicacid molecules of (a) or (b) in codon sequence due to the degeneracy ofthe genetic code, and (d) complements of (a), (b) or (c). The isolatednucleic acid molecules include isolated nucleic acid molecules that codefor a 2-O sulfatase which has an amino acid sequence set forth as SEQ IDNOs: 2 and 4.

The invention also includes degenerate nucleic acids which includealternative codons to those present in the native materials. Forexample, serine residues are encoded by the codons TCA, AGT, TCC, TCG,TCT and AGC. Each of the six codons is equivalent for the purposes ofencoding a serine residue. Thus, it will be apparent to one of ordinaryskill in the art that any of the serine-encoding nucleotide triplets maybe employed to direct the protein synthesis apparatus, in vitro or invivo, to incorporate a serine residue into an elongating 2-O sulfatase.Similarly, nucleotide sequence triplets which encode other amino acidresidues include, but are not limited to: CCA, CCC, CCG and CCT (prolinecodons); CGA, CGC, CGG, CGT, AGA and AGG (arginine codons); ACA, ACC,ACG and ACT (threonine codons); AAC and AAT (asparagine codons); andATA, ATC and ATT (isoleucine codons). Other amino acid residues may beencoded similarly by multiple nucleotide sequences. Thus, the inventionembraces degenerate nucleic acids that differ from the biologicallyisolated nucleic acids in codon sequence due to the degeneracy of thegenetic code.

The isolated nucleic acid molecules of the invention are also intendedto encompass homologs and alleles which can be identified byconventional techniques. Identification of human and other organismhomologs of 2-O sulfatase polypeptides will be familiar to those ofskill in the art. In general, nucleic acid hybridization is a suitablemethod for identification of homologous sequences of another species(e.g., human, cow, sheep), which correspond to a known sequence.Standard nucleic acid hybridization procedures can be used to identifyrelated nucleic acid sequences of selected percent identity. Forexample, one can construct a library of cDNAs reverse transcribed fromthe mRNA of a selected tissue and use the nucleic acids that encode a2-O sulfatase identified herein to screen the library for relatednucleotide sequences. The screening preferably is performed usinghigh-stringency conditions to identify those sequences that are closelyrelated by sequence identity. Nucleic acids so identified can betranslated into polypeptides and the polypeptides can be tested foractivity.

The term “stringent conditions” as used herein refers to parameters withwhich the art is familiar. Such parameters include salt, temperature,length of the probe, etc. The amount of resulting base mismatch uponhybridization can range from near 0% (“high stringency”) to about 30%(“low stringency”). Nucleic acid hybridization parameters may be foundin references that compile such methods, e.g. Molecular Cloning: ALaboratory Manual, J. Sambrook, et al., eds., Second Edition, ColdSpring Harbor Laboratory Press, Cold Spring Harbor, New York, 1989, orCurrent Protocols in Molecular Biology, F.M. Ausubel, et al., eds., JohnWiley&Sons, Inc., New York. One example of high-stringency conditions ishybridization at 650C in hybridization buffer (3.5×SSC, 0.02% Ficoll,0.02% polyvinyl pyrrolidone, 0.02% Bovine Serum Albumin, 2.5 mMNaH2PO4(pH7), 0.5% SDS, 2 mM EDTA). SSC is 0.15M sodium chloride/0.015Msodium citrate, pH7; SDS is sodium dodecyl sulphate; and EDTA isethylenediaminetetracetic acid. After hybridization, a membrane uponwhich the nucleic acid is transferred is washed, for example, in 2×SSCat room temperature and then at 0.1-0.5×SSC/0.1×SDS at temperatures upto 68° C.

The skilled artisan also is familiar with the methodology for screeningcells for expression of such molecules, which then are routinelyisolated, followed by isolation of the pertinent nucleic acid. Thus,homologs and alleles of the 2-O sulfatase of the invention, as well asnucleic acids encoding the same, may be obtained routinely, and theinvention is not intended to be limited to the specific sequencesdisclosed. It will be understood that the skilled artisan will be ableto manipulate the conditions in a manner to permit the clearidentification of homologs and alleles of the 2-O sulfatase nucleicacids of the invention. The skilled artisan also is familiar with themethodology for screening cells and libraries for expression of suchmolecules which then are routinely isolated, followed by isolation ofthe pertinent nucleic acid molecule and sequencing.

In general, homologs and alleles typically will share at least 90%nucleotide identity and/or at least 95% amino acid identity to thesequences of 2-O sulfatase nucleic acids and polypeptides, respectively,in some instances will share at least 95% nucleotide identity and/or atleast 97% amino acid identity, in other instances will share at least97% nucleotide identity and/or at least 98% amino acid identity, inother instances will share at least 99% nucleotide identity and/or atleast 99% amino acid identity, and in other instances will share atleast 99.5% nucleotide identity and/or at least 99.5% amino acididentity. The homology can be calculated using various, publiclyavailable software tools developed by NCBI (Bethesda, Md.) that can beobtained through the internet. Exemplary tools include the BLAST systemavailable from the website of the National Center for BiotechnologyInformation (NCBI) at the National Institutes of Health. Pairwise andClustalW alignments (BLOSUM30 matrix setting) as well as Kyte-Doolittlehydropathic analysis can be obtained using the MacVector sequenceanalysis software (Oxford Molecular Group). Watson-Crick complements ofthe foregoing nucleic acids also are embraced by the invention.

In screening for 2-O sulfatase related genes, such as homologs andalleles of 2-O sulfatase, a Southern blot may be performed using theforegoing conditions, together with a radioactive probe. After washingthe membrane to which the DNA is finally transferred, the membrane canbe placed against X-ray film or a phosphoimager plate to detect theradioactive signal.

The recombinantly produced 2-O sulfatase as provided herein exhibitedrobust, 2-O specific sulfatase activity. The success with expressing ahighly active 2-O sulfatase clearly validates our use of E. coli as arecombinant expression system for the large-scale production of activeenzyme. Therefore, active isolated 2-O sulfatase polypeptides (includingwhole proteins and partial proteins) are provided herein which includeisolated 2-O sulfatase polypeptides that have the amino acid sequence ofSEQ ID NO: 2 or SEQ ID NO: 4.

Polypeptides can be isolated from biological samples, and can also beexpressed recombinantly in a variety of prokaryotic and eukaryoticexpression systems, such as those described above, by constructing anexpression vector appropriate to the expression system, introducing theexpression vector into the expression system, and isolating therecombinantly expressed protein. Polypeptides can also be synthesizedchemically using well-established methods of peptide synthesis.

As used herein, “isolated polypeptide” means the polypeptide isseparated from its native environment and present in sufficient quantityto permit its identification or use. This means, for example: (i)selectively produced by expression cloning or (ii) purified as bychromatography or electrophoresis. Isolated proteins or polypeptides maybe, but need not be, substantially pure. Because an isolated polypeptidemay be admixed with a pharmaceutically acceptable carrier in apharmaceutical preparation, the polypeptide may comprise only a smallpercentage by weight of the preparation. The polypeptide is nonethelessisolated in that it has been separated from the substances with which itmay be associated in living systems, i.e., isolated from other proteins.

As used herein, the term “substantially pure” means that the proteinsare essentially free of other substances to an extent practical andappropriate for their intended use. In particular, the proteins aresufficiently pure and are sufficiently free from other biologicalconstituents of their hosts cells so as to be useful in, for example,protein sequencing, or producing pharmaceutical preparations. As usedherein, a “substantially pure 2-O sulfatase” is a preparation of 2-Osulfatase which has been isolated or synthesized and which is greaterthan about 90% free of contaminants. Preferably, the material is greaterthan 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or even greater than 99%free of contaminants. The degree of purity may be assessed by meansknown in the art. One method for assessing the purity of the materialmay be accomplished through the use of specific activity assays.

The cloned, full-length gene of the 2-O sulfatase encodes an openreading frame (ORF) of 468 amino acids (FIG. 2), with a predictedmolecular mass of 51.9 kDa. This theoretical molecular weight isapproximately 10 kDa less than the value reported in the literature(McLean, M. W., Bruce, J. S., Long, W. F., and Williamson, F. B. (1984)Eur J Biochem 145(3), 607-15). Based on its amino acid composition, theencoded protein is quite basic (theoretical pl of 8.75). A furtheranalysis of its primary amino acid sequence unequivocally places thisORF as a member of a larger sulfatase family. As members of a largeenzyme family, the sulfatases hydrolyze a wide array of sulfate esters(for a review, see (Parenti, G., Meroni, G., and Ballabio, A. (1997)Curr Opin Genet Dev 7(3), 386-91, von Figura, K., Schmidt, B., Selmer,T., and Dierks, T. (1998) Bioessays 20(6), 505-10)). Their respectivesubstrates include sulfated complex carbohydrates such as theglycosaminoglycans (GAGs), steroids, sphingolipids, xenobioticcompounds, and amino acids such as tyrosine. Additionally, many of theseenzymes are able to hydrolyze in vitro smaller synthetic substrates(e.g., 4-nitrophenyl sulfate and catechol sulfate). It is for thisreason that these enzymes are often generically described as“arylsulfatases” (even when their preferred in vivo substrate isill-defined). Despite their disparate substrate specificities, themembers of this enzyme family share both considerable structuralhomology and a common catalytic mechanism with one another (Waldow, A.,Schmidt, B., Dierks, T., von Bulow, R., and von Figura, K. (1999) J BiolChem 274(18), 12284-8).

The flavobacterial 2-O sulfatase possesses considerable sequencehomology to other bacterial (and non-bacterial) sulfatases, especiallywithin its amino terminus in which resides a highly conserved sulfatasedomain. This signature catalytic domain is readily identified by theconsensus sequence C/SXPXRXXXXS/TG (SEQ. ID NO: 6). The conservedcysteine (or less commonly serine) within this sulfatase motif is ofparticular functional importance as it is covalently modified to aL-Cα-formylglycine (L-2-amino-3-Oxo-propionic acid). The ubiquitousimportance of this chemical modification was first functionallyidentified by its relationship to the etiology of multiple sulfatasedeficiency (MSD), a genetically recessive disorder in which there is acomplete loss of sulfatase activity due to a lack of this criticalaldehyde (FGly) within the active site of all expressed sulfatases(Kolodny, E. H. a. F., A. L. (1995) in The Metabolic and Molecular Basesof Inherited Disease (Scriver, C. R., Beaudet, A. L., Sly, W. S., andValle, D., ed), pp. 2693-2741, McGraw-Hill, New York). We haveidentified the conserved sulfatase active site by sequence homologywhich we have found includes a cysteine and not a serine as the criticalamino acid predicted to be chemically modified as a formylglycine invivo. An empirical demonstration of this active-site aldehyde at thisposition is presented in Examples.

While the cloned flavobacterial sulfatase exhibits the highest sequencesimilarity to the bacterial arylsulfatases (especially the arylsulfatasefrom Pseudomonas aeruginosa), we point out that a limited homology ofthe 2-O sulfatase does extend to the mammalian glycosaminoglycansulfatases functioning in the lysosomal degradation pathway. As is thecase for the bacterial enzymes, this sequence homology is strongest inthe NH₂-terminus where the putative sulfatase domain resides. Among thehuman lysosomal enzymes, it is the galactosamine (N-acetyl)-6-sulfatesulfatase (chondroitin 6-O sulfatase) which exhibits the closestsimilarity with the flavobacterial 2-O sulfatase; the two enzymespossess approximately 26% identity when comparing their entire proteinsequences. There are also two functionally related lysosomal sulfataseswhich specifically hydrolyze the 2-OH position of uronic acid. Theseenzymes are the iduronate 2-sulfate sulfatase (IDS) (Bielicki, J.,Freeman, C., Clements, P. R., and Hopwood, J. J. (1990) Biochem J271(1), 75-86) and the glucuronic-2-sulfate sulfatase (Freeman, C., andHopwood, J. J. (1989) Biochem J 259(l), 209-16). The IDS andflavobacterial 2-O sulfatase exhibit only a limited sequence homology(less than 22% identity), however.

Both of these enzymes desulfate heparan sulfate, the iduronate-2-sulfatesulfatase (IDS) also acts on dermatan sulfate. Both enzymes possess anacidic pH optima for activity, a fact consistent with their locationwithin the lysosome. The two sulfatases initially exist as precursorswhich must be proteolytically processed for activity. The nativemolecular weight of the human IDS precursor has been reported in therange of 42 to 65 kDa (Bielicki, J., Freeman, C., Clements, P. R., andHopwood, J. J. (1990) Biochem J 271(1), 75-86), while its theoreticalmass based entirely on its amino acid composition is approximately 62kDa. As such, the mammalian lysosomal IDS is somewhat larger than itsflavobacterial counterpart, while also requiring substantialposttranslational modification for maximal enzyme activity. The acidicpH optima for the lysosomal enzymes would also appear to limit their invitro use for the determination of HSGAG composition, at least when usedin tandem with other flavobacterial HSGAG degrading enzymes such as theheparinases or the Δ 4,5 glycuronidase; these latter enzymes all possessa pH optima much closer to neutrality.

A homology-based structural model of the 2-O sulfatase active site wasconstructed using as a framework the available crystallographic data forthree highly related arylsulfatases. In this model, we have identifiedimportant structural parameters within the enzyme active site relevantto enzyme function, especially as relates to its substrate specificity(substrate binding and catalysis). By docking various disaccharidesubstrates, we were also able to make specific predictions concerningstructural determinants present within these potential substrates thatwould complement this unique active site architecture. Thesedeterminants included the position and number of sulfates present on theglucosamine, oligosaccharide chain length, the presence of a Δ 4,5unsaturated double bond, and the exolytic vs. endolytic potential of theenzyme. These predictions were-then tested against biochemical andkinetic data which largely validated our substrate specificitypredictions. Our modeling approach was further complementedexperimentally using aldehyde-specific chemical labeling, peptidemapping in tandem with mass spectrometry and site-directed mutagenesisto physically demonstrate the presence of a covalently modified cysteine(formyl glycine (FGly)) within the active site. This combinatorialapproach of structure modeling and biochemical studies has providedinsight into the molecular basis of enzyme function.

The crystal structures of two human lysosomal sulfatases,cerebroside-3-sulfate 3-sulfohydrolase (arylsulfatase A), (Lukatela, G.,Krauss, N., Theis, K., Selmer, T., Gieselmann, V., von Figura, K., andSaenger, W. (1998) Biochemistry 37(11), 3654-64, von Bulow, R., Schmidt,B., Dierks, T., von Figura, K., and Uson, I. (2001) J Mol Biol 305(2),269-77) N-acetylgalactosamine-4-sulfate 4-sulfohydrolase (arylsulfataseB) (Bond, C. S., Clements, P. R., Ashby, S. J., Collyer, C. A., Harrop,S. J., Hopwood, J. J., and Guss, J. M. (1997) Structure 5(2), 277-89),and a bacterial arylsulfatase from Pseudomonas aeruginosa (Boltes, I.,Czapinska, H., Kahnert, A., von Bulow, R., Dierks, T., Schmidt, B., vonFigura, K., Kertesz, M. A., and Uson, I. (2001) Structure (Camb) 9(6),483-91) have been solved. These three sulfatases share an identicalalkaline-phosphatase like structural fold (according to StructuralClassification of Proteins database (www.pdb.org)) comprised of a seriesof mixed parallel and antiparallel β strands flanked by long and short αhelices on either side (Lukatela, G., Krauss, N., Theis, K., Selmer, T.,Gieselmann, V., von Figura, K., and Saenger, W. (1998) Biochemistry37(11), 3654-64, Bond, C. S., Clements, P. R., Ashby, S. J., Collyer, C.A., Harrop, S. J., Hopwood, J. J., and Guss, J. M. (1997) Structure5(2), 277-89, Boltes, I., Czapinska, H., Kahnert, A., von Bulow, R.,Dierks, T., Schmidt, B., von Figura, K., Kertesz, M. A., and Uson, I.(2001) Structure (Camb) 9(6), 483-91, von Bulow, R., Schmidt, B.,Dierks, T., von Figura, K., and Uson, I. (2001) J Mol Biol 305(2),269-77). In addition to their common structural fold, these sulfatasestructures also possess a high degree of homology within theirrespective active sites, especially in the region localized around themodified cysteine (FGly). Taken together, these crystal structurespresent a clear and consistent description of conserved active siteresidues at least as it relates to a likewise conserved mechanism ofsulfate ester hydrolysis. At the same time, this strong structuralhomology is somewhat surprising considering that at least two of thesesulfatases act on notably different substrates, e.g., sulfatedsphingolipid vs. sulfated glycosaminoglycan (GAG).

It was discovered that 2-O sulfatase has a relatively high cysteinecontent. Apart from the catalytic cysteine at position 82, none of theremaining seven cysteines appeared to be highly conserved among othermembers of the sulfatase family. Enzyme activity was not inhibited withthe addition of DTNB (Ellman's reagent) or DTT. This general lack ofinhibition by these two cysteine-reactive agents suggests at least twoprobabilities. First, the 2-O sulfatase does not require intramoleculardisulfide linkages to critically stabilize a catalytically activeconformation. Second, free sulfhydryls are not directly participating incatalysis. It is possible, however, that a few of these cysteines areburied and therefore not accessible to sulfhydryl exchange. At leastfive of the eight cysteines, however, do react with DTNB undernondenaturing conditions. This latter fact suggests an alternate rolefor these solvent-accessible cysteines (along with specific histidines)ie., metal-coordinating thiolates. Comparison between the 2-O sulfataseand alkaline phosphatase reveals that these enzymes are esterases withsimilar catalytic mechanisms, including the presumptive formation of acovalent intermediate. The two hydrolytic enzymes also possessstructurally related domains, in particular, a highly superimposibleactive site that includes a divalent metal binding pocket. In the caseof alkaline phosphatase, it is zinc rather than calcium (or Mg⁺²) thatis coordinated within this pocket.

The 2-O sulfatase possesses 67 basic amino acids, including thecatalytic histidine at position 136, a proximal lysine at position 134and an invariant arginine at position 86 found within the definingsulfatase consensus sequence. Moreover, crystal structures of the activesite of related sulfatases each clearly show at least four basicresidues participating in catalysis which was also found in our homologymodel. A masking of these important charges by exogenous ions wouldinterfere with their catalytic function.

Of the 8 histidines present in the flavobacterial 2-O sulfatase, H136 isinvariantly conserved among the structurally related bacterialsulfatases examined. For each of these enzymes, this highly conservedhistidine is found within a putative consensus sequence GKWHX (SEQ. IDNO: 7) (where X is a hydrophobic amino acid). Other conserved histidinesinclude His 296 and His 303. Catalytically important histidines havebeen observed within the active site of several sulfatase crystalstructures including human lysosomal N-acetylgalactosamine-4 sulfatase(arylsulfatase B) (Bond, C. S., Clements, P. R., Ashby, S. J., Collyer,C. A., Harrop, S. J., Hopwood, J. J., and Guss, J. M. (1997) Structure5(2), 277-89) and arylsulfatase A (Lukatela, G., Krauss, N., Theis, K.,Selmer, T., Gieselmann, V., von Figura, K., and Saenger, W. (1998)Biochemistry 37(11), 3654-64) as well as the arysulfatase fromPseudomonas aeriginosa (Boltes, I., Czapinska, H., Kahnert, A., vonBulow, R., Dierks, T., Schmidt, B., von Figura, K., Kertesz, M. A., andUson, I. (2001) Structure (Camb) 9(6), 483-91) to which theflavobacterial 2-O sulfatase appears to be most closely related. In thelatter case, His211 appears to hydrogen bond with the sulfate oxygen(O4) contributing perhaps to proper sulfate coordination. Additionally,the Nδ1 of His 115 of P. aeruginosa (His 242 in the human 4-S sulfatase)is within hydrogen bonding distance to the Oγ2 of the catalyticformylglycine. The presence of His 136 in the active site and itsparticipation in catalysis is strongly supported by our homologystudies.

The flavobacterial 2-O sulfatase possesses 52 acidic amino acids,several of which are highly conserved (e.g., Asp 42, Asp 269, Asp 286,Asp 295, and Asp 342). Interestingly, four acidic side chains are alsofound in a consensus active site also observed in known crystalstructures. In this snapshot, these four carboxylates appear tocoordinate a divalent metal ion (typically calcium). This divalent metalin turn coordinates with the formylglycine hydroxylate and possibly theOγ1 group of the sulfate.

Based on the understanding of the important residues involved in thefunction of 2-O sulfatase, the invention also embraces functionalvariants. As used herein, a “functional variant” of a 2-O sulfatasepolypeptide is a polypeptide which contains one or more modifications tothe primary amino acid sequence of a 2-O sulfatase polypeptide. Thepolypeptide can contain 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12 ,13 ,14,15, 16, 17, 18, 19, 20, 25, 30, 35, 40, 50 or more amino acidmodifications. These modifications are intended to encompassmodifications that result in a 2-O sulfatase with altered activityrelative to the native 2-O sulfatase but also include modifications thatdo not result in altered activity relative to the native enzyme. Theterm “native” as used herein refers to the 2-O sulfatase as it would befound in nature. Modifications which create a 2-O sulfatase polypeptidefunctional variant are typically made to the nucleic acid which encodesthe 2-O sulfatase polypeptide, and can include deletions, pointmutations, truncations, amino acid substitutions and addition of aminoacids or non-amino acid moieties to: 1) enhance a property of a 2-Osulfatase polypeptide, such as protein stability in an expression systemor the stability of protein-protein binding; 2) provide a novel activityor property to a 2-O sulfatase polypeptide, such as addition of adetectable moiety; or 3) to provide equivalent or better interactionwith other molecules (e.g., heparin). Alternatively, modifications canbe made directly to the polypeptide, such as by cleavage, addition of alinker molecule, addition of a detectable moiety, such as biotin,addition of a fatty acid, and the like. Modifications also embracefusion proteins comprising all or part of the 2-O sulfatase amino acidsequence. One of skill in the art will be familiar with methods forpredicting the effect on protein conforrmation of a change in proteinsequence, and can thus “design” a functional variant 2-O sulfatasepolypeptide according to known methods. One example of such a method isdescribed by Dahiyat and Mayo in Science 278:82-87, 1997, wherebyproteins can be designed de novo. The method can be applied to a knownprotein to vary only a portion of the polypeptide sequence. By applyingthe computational methods of Dahiyat and Mayo, specific variants of apolypeptide can be proposed and tested to determine whether the variantretains a desired conformation.

Functional variants can include 2-O sulfatase polypeptides which aremodified specifically to alter a feature of the polypeptide unrelated toits physiological activity. For example, cysteine residues can besubstituted or deleted to prevent unwanted disulfide linkages.Similarly, certain amino acids can be changed to enhance expression of a2-O sulfatase polypeptide by eliminating proteolysis by proteases in anexpression system (e.g., dibasic amino acid residues in yeast expressionsystems in which KEX2 protease activity is present). Functionalvariants, therefore, can also include variant 2-O sulfatase thatmaintain the same enzymatic function as the native 2-O sulfatase butinclude some modification to the amino acid sequence that does not alternative enzyme activity. These modifications include conservative aminoacid substitutions as well as non-conservative amino acid substitutionsthat are remote from the binding and catalytic sites of the enzyme.

Mutations of a nucleic acid which encodes a 2-O sulfatase polypeptidepreferably preserve the amino acid reading frame of the coding sequence,and preferably do not create regions in the nucleic acid which arelikely to hybridize to form secondary structures, such as hairpins orloops, which can be deleterious to expression of the variantpolypeptide.

Mutations can be made by selecting an amino acid substitution, or byrandom mutagenesis of a selected site in a nucleic acid which encodesthe polypeptide. Variant polypeptides are then expressed and tested forone or more activities to determine which mutation provides a variantpolypeptide with the desired properties. Further mutations can be madeto variants (or to-non-variant 2-O sulfatase polypeptides) which aresilent as to the amino acid sequence of the polypeptide, but whichprovide preferred codons for translation in a particular host. Thepreferred codons for translation of a nucleic acid in, e.g., E. coli,are well known to those of ordinary skill in the art. Still othermutations can be made to the noncoding sequences of a 2-O sulfatase geneor cDNA clone to enhance expression of the polypeptide.

In the description that follows, reference will be made to the aminoacid residues and residue positions of native 2-O sulfatase disclosed inSEQ ID NO: 1. In particular, residues and residue positions will bereferred to as “corresponding to” a particular residue or residueposition of 2-O sulfatase. As will be obvious to one of ordinary skillin the art, these positions are relative and, therefore, insertions ordeletions of one or more residues would have the effect of altering thenumbering of downstream residues. In particular, N-terminal insertionsor deletions would alter the numbering of all subsequent residues.Therefore, as used herein, a residue in a recombinant modifiedheparinase will be referred to as “corresponding to” a residue of thefull 2-O sulfatase if, using standard sequence comparison programs, theywould be aligned. Many such sequence alignment programs are nowavailable to one of ordinary skill in the art and their use in sequencecomparisons has become standard (e.g., “LALIGN” available via theInternet at http://phaedra.crbm.cnrs-mop.fr/fasta/lalign-query.html). Asused herein, this convention of referring to the positions of residuesof the recombinant modified heparinases by their corresponding 2-Osulfatase residues shall extend not only to embodiments includingN-terminal insertions or deletions but also to internal insertions ordeletions (e.g, insertions or deletions in “loop” regions).

One type of amino acid substitution is referred to as a “conservativesubstitution.” As used herein, a “conservative amino acid substitution”or “conservative substitution” refers to an amino acid substitution inwhich the substituted amino acid residue is of similar charge as thereplaced residue and is of similar or smaller size than the replacedresidue. Conservative substitutions of amino acids include substitutionsmade amongst amino acids within the following groups: (a) the smallnon-polar amino acids, A, M, I, L, and V; (b) the small polar aminoacids, G, S, T and C; (c) the amido amino acids, Q and N; (d) thearomatic amino acids, F, Y and W; (e) the basic amino acids, K, R and H;and (f) the acidic amino acids, E and D. Substitutions which are chargeneutral and which replace a residue with a smaller residue may also beconsidered “conservative substitutions” even if the residues are indifferent groups (e.g., replacement of phenylalanine with the smallerisoleucine). The term “conservative amino acid substitution” also refersto the use of amino acid analogs.

Methods for making amino acid substitutions, additions or deletions arewell known in the art. The terms “conservative substitution”,“non-conservative substitutions”, “non-polar amino acids”, “polar aminoacids”, and “acidic amino acids” are all used consistently with theprior art terminology. Each of these terms is well-known in the art andhas been extensively described in numerous publications, includingstandard biochemistry text books, such as “Biochemistry” by GeoffreyZubay, Addison-Wesley Publishing Co., 1986 edition, which describesconservative and non-conservative substitutions, and properties of aminoacids which lead to their definition as polar, non-polar or acidic.

Even when it is difficult to predict the exact effect of a substitutionin advance of doing so, one skilled in the art will appreciate that theeffect can be evaluated by routine screening assays, preferably thebiological assays described herein. Modifications of peptide propertiesincluding thermal stability, enzymatic activity, hydrophobicity,susceptibility to proteolytic degradation or the tendency to aggregatewith carriers or into multimers are assayed by methods well known to theordinarily skilled artisan. For additional detailed description ofprotein chemistry and structure, see Schulz, G. E. et al., Principles ofProtein Structure, Springer-Verlag, New York, 1979, and Creighton, T.E., Proteins: Structure and Molecular Principles, W. H. Freeman & Co.,San Francisco, 1984.

Additionally, some of the amino acid substitutions are non-conservativesubstitutions. In certain embodiments where the substitution is remotefrom the active or binding sites, the non-conservative substitutions areeasily tolerated provided that they preserve a tertiary structurecharacteristic of, or similar to, native 2-O sulfatase, therebypreserving the active and binding sites. Non-conservative substitutions,such as between, rather than within, the above groups (or two otheramino acid groups not shown above), which will differ more significantlyin their effect on maintaining (a) the structure of the peptide backbonein the area of the substitution (b) the charge or hydrophobicity of themolecule at the target site, or (c) the bulk of the side chain.

Nearly every active, recombinantly expressed sulfatase reported in theliterature possesses a cysteine (and not a serine) within the activesite sequence C/SXPXRXXXXS/TG (SEQ. ID NO: 6) (Lukatela, G., Krauss, N.,Theis, K., Selmer, T., Gieselmann, V., von Figura, K., and Saenger, W.(1998) Biochemistry 37(11), 3654-64). It seemed likely, therefore, thata cysteine-specific modifying machinery functionally exists in E. coli.This idea was supported by our initial attempts to produce a recombinantcysteine→serine 2-O sulfatase variant which led to the production ofinsoluble protein when expressed in E. coli. We note that the E. coligenome encodes for at least three different putative sulfatase genes inaddition to the atsB gene which, by homology, has been proposed toencode for this cysteine-specific modifying activity. All of these genesare located as a cluster within the bacterial chromosome (Kertesz, M. A.(2000) FEMS Microbiol Rev 24(2), 135-75). It would appear, however, thatthe E. coli sulfatase genes are normally cryptic. At the very least, E.coli lacks the specific enzymes for desulfating heparin/heparan sulfateglycosaminoglycans, but the bacterium fortuitously provides thenecessary enzymology to effectively modify select heterologoussulfatases such as the 2-O sulfatase. Therefore, the 2-O sulfatases asdescribed herein can be produced recombinantly in E. coli. However, therecombinant production of the 2-O sulfatases provided are not limited totheir expression in E. coli. The 2-O sulfatases can also berecombinantly produced in other expression systems described below.

The 2-O sulfatases, can be recombinantly produced using a vectorincluding a coding sequence operably joined to one or more regulatorysequences. As used herein, a coding sequence and regulatory sequencesare said to be “operably joined” when they are covalently linked in sucha way as to place the expression or transcription of the coding sequenceunder the influence or control of the regulatory sequences. If it isdesired that the coding sequences be translated into a functionalprotein the coding sequences are operably joined to regulatorysequences. Two DNA sequences are said to be operably joined if inductionof a promoter in the 5′ regulatory sequences results in thetranscription of the coding sequence and if the nature of the linkagebetween the two DNA sequences does not (1) result in the introduction ofa frame-shift mutation, (2) interfere with the ability of the promoterregion to direct the transcription of the coding sequences, or (3)interfere with the ability of the corresponding RNA transcript to betranslated into a protein. Thus, a promoter region would be operablyjoined to a coding sequence if the promoter region were capable ofeffecting transcription of that DNA sequence such that the resultingtranscript might be translated into the desired protein or polypeptide.

The precise nature of the regulatory sequences needed for geneexpression may vary between species or cell types, but shall in generalinclude, as necessary, 5′ non-transcribing and 5′ non-translatingsequences involved with initiation of transcription and translationrespectively, such as a TATA box, capping sequence, CAAT sequence, andthe like. Especially, such 5′ non-transcribing regulatory sequences willinclude a promoter region which includes a promoter sequence fortranscriptional control of the operably joined gene. Promoters may beconstitutive or inducible. Regulatory sequences may also includeenhancer sequences or upstream activator sequences, as desired.

As used herein, a “vector” may be any of a number of nucleic acids intowhich a desired sequence may be inserted by restriction and ligation fortransport between different genetic environments or for expression in ahost cell. Vectors are typically composed of DNA although RNA vectorsare also available. Vectors include, but are not limited to, plasmidsand phagemids. A cloning vector is one which is able to replicate in ahost cell, and which is further characterized by one or moreendonuclease restriction sites at which the vector may be cut in adeterminable fashion and into which a desired DNA sequence may beligated such that the new recombinant vector retains its ability toreplicate in the host cell. In the case of plasmids, replication of thedesired sequence may occur many times as the plasmid increases in copynumber within the host bacterium, or just a single time per host as thehost reproduces by mitosis. In the case of phage, replication may occuractively during a lytic phase or passively during a lysogenic phase. Anexpression vector is one into which a desired DNA sequence may beinserted by restriction and ligation such that it is operably joined toregulatory sequences and may be expressed as an RNA transcript. Vectorsmay further contain one or more marker sequences suitable for use in theidentification of cells which have or have not been transformed ortransfected with the vector. Markers include, for example, genesencoding proteins which increase or decrease either resistance orsensitivity to antibiotics or other compounds, genes which encodeenzymes whose activities are detectable by standard assays known in theart (e.g., β-galactosidase or alkaline phosphatase), and genes whichvisibly affect the phenotype of transformed or transfected cells, hosts,colonies or plaques. Preferred vectors are those capable of autonomousreplication and expression of the structural gene products present inthe DNA segments to which they are operably joined.

For prokaryotic systems, plasmid vectors that contain replication sitesand control sequences derived from a species compatible with the hostmay be used. Examples of suitable plasmid vectors include pBR322, pUC18,pUC19 and the like; suitable phage or bacteriophage vectors includeλgt10, λgt11 and the like; and suitable virus vectors include pMAM-neo,PKRC and the like. Preferably, the selected vector of the presentinvention has the capacity to autonomously replicate in the selectedhost cell. Useful prokaryotic hosts include bacteria, in addition to E.coli, Flavobacterium heparinum, Bacillus, Streptomyces, Pseudomonas,Salmonella, Serratia, and the like.

To express the 2-O sulfatase of the invention in a prokaryotic cell, itis desirable to operably join the nucleic acid sequence of a 2-Osulfatase of the invention to a functional prokaryotic promoter. Suchpromoter may be either constitutive or, more preferably, regulatable(i.e., inducible or derepressible). Examples of constitutive promotersinclude the int promoter of bacteriophage λ, the bla promoter of theβ-lactamase gene sequence of pBR322, and the CAT promoter of thechloramphenicol acetyl transferase gene sequence of pPR325, and thelike. Examples of inducible prokaryotic promoters include the majorright and left promoters of bacteriophage λ (P_(L) and P_(R)), the trp,recA, lacZ, lacI, and gal promoters of E. coli, the α-amylase (Ulmanenet al., J. Bacteriol. 162:176-182 (1985)) and the ζ-28-specificpromoters of B. subtilis (Gilman et al., Gene sequence 32:11-20 (1984)),the promoters of the bacteriophages of Bacillus (Gryczan, In: TheMolecular Biology of the Bacilli, Academic Press, Inc., NY (1982)), andStreptomyces promoters (Ward et al., Mol. Gen. Genet. 203:468-478(1986)).

Prokaryotic promoters are reviewed by Glick (J. Ind. Microbiol.1:277-282 (1987)); Cenatiempo (Biochimie 68:505-516 (1986)); andGottesman (Ann. Rev. Genet. 18:415-442 (1984)).

Proper expression in a prokaryotic cell also requires the presence of aribosome binding site upstream of the encoding sequence. Such ribosomebinding sites are disclosed, for example, by Gold et al. (Ann. Rev.Microbiol. 35:365-404 (1981)).

Because prokaryotic cells may not produce the 2-O sulfatase of theinvention with normal eukaryotic glycosylation, expression of the 2-Osulfatase of the invention of the eukaryotic hosts is useful whenglycosylation is desired. Preferred eukaryotic hosts include, forexample, yeast, fungi, insect cells, and mammalian cells, either in vivoor in tissue culture. Mammalian cells which may be useful as hostsinclude HeLa cells, cells of fibroblast origin such as VERO or CHO-K1,or cells of lymphoid origin, such as the hybridoma SP2/0-AG14 or themyeloma P3×63Sg8, and their derivatives. Preferred mammalian host cellsinclude SP2/0 and J558L, as well as neuroblastoma cell lines such as IMR332 that may provide better capacities for correct post-translationalprocessing. Embryonic cells and mature cells of a transplantable organalso are useful according to some aspects of the invention.

In addition, plant cells are also available as hosts, and controlsequences compatible with plant cells are available, such as thenopaline synthase promoter and polyadenylation signal sequences.

Another preferred host is an insect cell, for example in Drosophilalarvae. Using insect cells as hosts, the Drosophila alcoholdehydrogenase promoter can be used (Rubin, Science 240:1453-1459(1988)). Alternatively, baculovirus vectors can be engineered to expresslarge amounts of the 2-O sulfatase of the invention in insect cells(Jasny, Science 238:1653 (1987); Miller et al., In: Genetic Engineering(1986), Setlow, J. K., et al., eds., Plenum, Vol. 8, pp. 277-297).

Any of a series of yeast gene sequence expression systems whichincorporate promoter and termination elements from the genes coding forglycolytic enzymes and which are produced in large quantities when theyeast are grown in media rich in glucose may also be utilized. Knownglycolytic gene sequences can also provide very efficienttranscriptional control signals. Yeast provide substantial advantages inthat they can also carry out post-translational peptide modifications. Anumber of recombinant DNA strategies exist which utilize strong promotersequences and high copy number plasmids which can be utilized forproduction of the desired proteins in yeast. Yeast recognize leadersequences on cloned mammalian gene sequence products and secretepeptides bearing leader sequences (i.e., pre-peptides).

A wide variety of transcriptional and translational regulatory sequencesmay be employed, depending upon the nature of the host. Thetranscriptional and translational regulatory signals may be derived fromviral sources, such as adenovirus, bovine papilloma virus, simian virus,or the like, where the regulatory signals are associated with aparticular gene sequence which has a high level of expression.Alternatively, promoters from mammalian expression products, such asactin, collagen, myosin, and the like, may be employed. Transcriptionalinitiation regulatory signals may be selected which allow for repressionor activation, so that expression of the gene sequences can bemodulated. Of interest are regulatory signals that aretemperature-sensitive so that by varying the temperature, expression canbe repressed or initiated, or which are subject to chemical (such asmetabolite) regulation.

As discussed above, expression of the 2-O sulfatase of the invention ineukaryotic hosts requires the use of eukaryotic regulatory regions. Suchregions will, in general, include a promoter region sufficient to directthe initiation of RNA synthesis. Preferred eukaryotic promoters include,for example, the promoter of the mouse metallothionein I gene sequence(Hamer et al., J. Mol. Appl. Gen. 1:273-288 (1982)); the TK promoter ofHerpes virus (McKnight, Cell 31:355-365 (1982)); the SV40 early promoter(Benoist et al., Nature (London) 290:304-310 (1981)); the yeast gal4gene sequence promoter (Johnston et al., Proc. Natl. Acad. Sci. (USA)79:6971-6975 (1982); Silver et al., Proc. Natl. Acad. Sci. (USA)81:5951-5955 (1984)).

As is widely known, translation of eukaryotic mRNA is initiated at thecodon which encodes the first methionine. For this reason, it ispreferable to ensure that the linkage between a eukaryotic promoter anda DNA sequence which encodes the 2-O sulfatase of the invention does notcontain any intervening codons which are capable of encoding amethionine (i.e., AUG). The presence of such codons results either inthe formation of a fusion protein (if the AUG codon is in the samereading frame as the 2-O sulfatase of the invention coding sequence) ora frame-shift mutation (if the AUG codon is not in the same readingframe as the 2-O sulfatase of the invention coding sequence).

In one embodiment, a vector is employed which is capable of integratingthe desired gene sequences into the host cell chromosome. Cells whichhave stably integrated the introduced DNA into their chromosomes can beselected by also introducing one or more markers which allow forselection of host cells which contain the expression vector. The markermay, for example, provide for prototrophy to an auxotrophic host or mayconfer biocide resistance to, e.g., antibiotics, heavy metals, or thelike. The selectable marker gene sequence can either be directly linkedto the DNA gene sequences to be expressed, or introduced into the samecell by co-transfection. Additional elements may also be needed foroptimal synthesis of the 2-O sulfatase mRNA. These elements may includesplice signals, as well as transcription promoters, enhancers, andtermination signals. cDNA expression vectors incorporating such elementsinclude those described by Okayama, Molec. Cell. Biol. 3:280 (1983).

In another embodiment, the introduced sequence will be incorporated intoa plasmid or viral vector capable of autonomous replication in therecipient host. Any of a wide variety of vectors may be employed forthis purpose. Factors of importance in selecting a particular plasmid orviral vector include: the ease with which recipient cells that containthe vector may be recognized and selected from those recipient cellswhich do not contain the vector; the number of copies of the vectorwhich are desired in a particular host; and whether it is desirable tobe able to “shuttle” the vector between host cells of different species.Preferred prokaryotic vectors include plasmids such as those capable ofreplication in E. coli (such as, for example, pBR322, ColEl, pSC101,pACYC 184, and πVX). Such plasmids are, for example, disclosed bySambrook, et al. (Molecular Cloning: A Laboratory Manual, secondedition, edited by Sambrook, Fritsch, & Maniatis, Cold Spring HarborLaboratory, 1989)). Bacillus plasmids include pC194, pC221, pT127, andthe like. Such plasmids are disclosed by Gryczan (In: The MolecularBiology of the Bacilli, Academic Press, NY (1982), pp. 307-329).Suitable Streptomyces plasmids include pIJ101 (Kendall et al., J.Bacteriol. 169:4177-4183 (1987)), and streptomyces bacteriophages suchas φC31 (Chater et al., In: Sixth International Symposium onActinomycetales Biology, Akademiai Kaido, Budapest, Hungary (1986), pp.45-54). Pseudomonas plasmids are reviewed by John et al. (Rev. Infect.Dis. 8:693-704 (1986)), and Izaki (Jpn. J. Bacteriol. 33:729-742(1978)).

Preferred eukaryotic plasmids include, for example, BPV, EBV, SV40,2-micron circle, and the like, or their derivatives. Such plasmids arewell known in the art (Botstein et al., Miami Wntr. Symp. 19:265-274(1982); Broach, In: The Molecular Biology of the Yeast Saccharomyces:Life Cycle and Inheritance, Cold Spring Harbor Laboratory, Cold SpringHarbor, N.Y., p. 445-470 (1981); Broach, Cell 28:203-204 (1982); Bollonet al., J. Clin. Hematol. Oncol. 10:39-48 (1980); Maniatis, In: CellBiology: A Comprehensive Treatise, Vol. 3, Gene Sequence Expression,Academic Press, NY, pp. 563-608 (1980)). Other preferred eukaryoticvectors are viral vectors. For example, and not by way of limitation,the pox virus, herpes virus, adenovirus and various retroviruses may beemployed. The viral vectors may include either DNA or RNA viruses tocause expression of the insert DNA or insert RNA.

Once the vector or DNA sequence containing the construct(s) has beenprepared for expression, the DNA construct(s) may be introduced into anappropriate host cell by any of a variety of suitable means, i.e.,transformation, transfection, conjugation, protoplast fusion,electroporation, calcium phosphate-precipitation, direct microinjection,and the like. Additionally, DNA or RNA encoding the 2-O sulfatase of theinvention may be directly injected into cells or may be impelled throughcell membranes after being adhered to microparticles. After theintroduction of the vector, recipient cells are grown in a selectivemedium, which selects for the growth of vector-containing cells.Expression of the cloned gene sequence(s) results in the production ofthe 2-O sulfatase of the invention. This can take place in thetransformed cells as such, or following the induction of these cells todifferentiate (for example, by administration of bromodeoxyuracil toneuroblastoma cells or the like).

One of skill in the art may also substitute appropriate codons toproduce the desired amino acid substitutions in SEQ ID NOs: 2 or 4 bystandard site-directed mutagenesis techniques. One may also use anysequence which differs from the nucleic acid equivalents of SEQ ID NO: 2or 4 only due to the degeneracy of the genetic code as the startingpoint for site directed mutagenesis. The mutated nucleic acid sequencemay then be ligated into an appropriate expression vector and expressedin a host such as E. coli.

Our initial assessment of 2-O sulfatase activity was based upon the useof a few select unsaturated heparin disaccharide substrates. Desulfationwas unequivocally specific for the 2-O position (FIG. 5). This substratediscrimination was based on the extent of sulfation and largelymanifested as a K_(m) effect. In particular, the presence of a 6-Osulfate on the adjoining glucosamine conferred a significantly lowerK_(m) relative to its counterpart lacking such a sulfate ester. In termsof catalytic efficiency, the trisulfated disaccharide (ΔU_(2S)H_(NS,6S))was the more efficient substrate whereas the mono-sulfated disaccharide(ΔU_(2S)H_(NAc)) was less efficient.

The 2-O sulfated chondroitin disaccharide ΔU_(2S)Gal_(NAc,6S), however,was also, albeit neglibly, hydrolyzed under the same kinetic conditions.The enzyme did desulfate this disaccharide to an appreciable extent,however, under reaction conditions involving a 4× higher enzymeconcentration and a longer incubation time. Under these conditions,approximately 40% of the substrate was desulfated over a 20 minuteperiod. In contrast, less than 10% of chondroitin disaccharideΔU_(2S)Gal_(NAc,4S) was hydrolyzed during the same time period. Underexhaustive conditions, both chondroitin disaccharides were greater than95% desulfated at the 2-O position. The apparent kinetic discriminationpoints to an underlying structural determinant, namely a preference forglucosamine sulfated at the 6-OH and 2N positions.

In addition, examination of the biochemical conditions for optimalenzymatic activity yielded several observations. First, 2-O sulfataseactivity exhibited a pH profile with a narrower pH range (6.0-7.0) inwhich the enzyme was most active. The enzyme exhibited maximal catalyticefficiency at pH 6.5 with essentially no activity observed at theoutlying pH values of 5 and 8 (FIG. 6, Panel (A)). A sharply defined pHoptima of 6.5 implicates a catalytic role of one or more histidines.Second, the observed NaCl titration profile (FIG. 6, Panel (B))demonstrates a clearly inhibitory effect of ionic strength on sulfataseactivity, even at relatively low NaCl concentrations. That is, while 50%inhibition occurred in the presence of approximately 200 mM NaCl, even100 mM NaCl was slightly inhibitory to 2-O sulfatase activity. This is arather sharp activity transition for both the Δ 4,5 glycuronidase andother recombinantly expressed F. heparinum GAG degrading enzymes. Thecorrelation between activity and the ionic buffer composition isreasonable, given the anionic character of the saccharide substratesconferred by both the presence of sulfates and the uronic acidcarboxylates within each disaccharide unit. For the 2-O sulfatase inparticular, charge interactions between basic side chains and thesulfate oxygen anion may be involved in substrate orientation.

The results described herein suggest that the 2-O sulfatase activity isupstream from the hydrolysis of the unsaturated uronic acid by the Δ 4,5glycuronidase. This scenario would also make the 2-O sulfatase aso-called “early” enzyme in the HSGAG degradation pathway that occurs invivo. The substrate-product correlation between the 2-O sulfatase andthe Δ 4,5 glycuronidase has been demonstrated with the two experimentssummarized in FIGS. 7 and 8. FIG. 8 in particular demonstrates how thesetwo enzymes (along with the heparinases) can be used in tandem asanalytical tools for HSGAG compositional analyses. The results havedemonstrated the utility of the sulfatase as a tool for probing HSGAGcomposition, especially when the enzyme is used in tandem with the Δ 4,5glycuronidase.

The present invention provides for the use of 2-O sulfatase as anenzymatic tool due to its substrate specificity and specific activity.As described herein, it was found that the activity of the cloned enzymeis not compromised by its recombinant expression in E. coli. The “native2-O sulfatase specific activity” is the measure of enzymatic activity ofthe native 2-O sulfatase obtained from cell lysates of F. heparinum alsodescribed in the Examples below. Therefore, based on the disclosureprovided herein, those of ordinary skill in the art will be able toidentify other 2-O sulfatases having altered enzymatic activity withrespect to the native 2-O sulfatase such as functional variants.

The term “specific activity” as used herein refers to the enzymaticactivity of a preparation of 2-O sulfatase. In general, it is preferredthat the substantially pure and/or isolated 2-O sulfatase preparationsof the invention have a specific activity of at least about 7 nanomolesof substrate (DiS) hydrolized per minute per microgram of enzyme. Italso generally more preferred that the substantially pure and/orisolated 2-O sulfatase preparations of the invention have a specificactivity of at least about 40 nanomoles of substrate (DiS) hydrolizedper minute per microgram of enzyme. As provided herein, the recombinant2-O sulfatase purified by (nickel chromatography with the histidine tag)was found to have an about six-fold higher specific activity than native2-O sulfatase. The recombinant 2-O sulfatase without the histidine tagwas found to have an about ten-fold higher specific activity than thenative 2-O sulfatase. Therefore, in one aspect of the inventionpreparations of 2-O sulfatase with about a 5-, 6-, 7-, 8-, 9-, 10-, 11-,12-, 13-, 14-, 15-, 20-, 25-, and 30-fold specific activity areprovided.

The invention, therefore, provides for the degradation ofglycosaminoglycans using the 2-O sulfatase described herein. The 2-Osulfatase of the invention may be used to specifically degrade an HSGAGby contacting the HSGAG substrate with the 2-O sulfatase of theinvention. The invention is useful in a variety of in vitro, in vivo andex vivo methods in which it is useful to degrade HSGAGs.

As used herein the terms “HSGAG” and “glycosaminoglycan” and “GAG” areused interchangeably to refer to a family of molecules havingheparin-like/heparan sulfate-like structures and properties. Thesemolecules include but are not limited to low molecular weight heparin(LMWH), heparin, biotechnologically prepared heparin, chemicallymodified heparin, synthetic heparin, and heparan sulfate. The term“biotechnological heparin” encompasses heparin that is prepared fromnatural sources of polysaccharides which have been chemically modifiedand is described for example in Razi et al., Bioche. J. Jul. 15,1995;309 (Pt 2): 465-72. Chemically modified heparin is described inYates et al., Carbohydrate Res Nov. 20, 1996;294:15-27, and is known tothose of skill in the art. Synthetic heparin is well known to those ofskill in the art and is described in Petitou, M. et al., Bioorg Med ChemLett. Apr. 19, 1999;9(8):1161-6.

Analysis of a sample of glycosaminoglycans is also possible with 2-Osulfatase alone or in conjunction with other enzymes. Other HSGAGdegrading enzymes include but are not limited to heparinase-I,heparinase-II , heparinase-III, Δ 4, 5 glycuronidase, other sulfatases,modified versions of the enzymes, variants and functionally activefragments thereof. In particular, 2-O sulfatase can be used subsequentto or concomitantly with a heparinase to degrade a glycosaminoglycan. Inaddition 2-O sulfatase may be used prior to and also concomitantly withΔ 4, 5 glycuronidase.

The methods that may be used to test the specific activity of 2-Osulfatase of the present invention are known in the art, e.g., thosedescribed in the Examples. These methods may also be used to assess thefunction of variants and functionally active fragments of 2-O sulfatase.The k_(cat) value may be determined using any enzymatic activity assayto assess the activity of a 2-O sulfatase enzyme. Several such assaysare well-known in the art. For instance, an assay for measuring k_(cat)is described in Ernst, S. E., Venkataraman, G., Winkler, S., Godavarti,R., Langer, R., Cooney, C. and Sasisekharan. R. (1996) Biochem. J. 315,589-597. Therefore, based on the disclosure provided herein, those ofordinary skill in the art will be able to identify other 2-O sulfatasemolecules having enzymatic activity that is similar to or altered incomparison with the native 2-O sulfatase molecule such as 2-O sulfatasefunctional variants.

Due to the activity of 2-O sulfatase on glycosaminoglycans, the productprofile produced by a 2-O sulfatase may be determined by any methodknown in the art for examining the type or quantity of degradationproducts produced by 2-O sulfatase alone or in combination with otherenzymes. One of skill in the art will also recognize that the 2-Osulfatase may also be used to assess the purity of glycosaminoglycans ina sample. One preferred method for determining the type and quantity ofproduct is described in Rhomberg, A. J. et al., PNAS, v. 95, p.4176-4181, (April 1998), which is hereby incorporated in its entirety byreference. The method disclosed in the Rhomberg reference utilizes acombination of mass spectrometry and capillary electrophoretictechniques to identify the enzymatic products produced by heparinase.The Rhomberg study utilizes heparinase to degrade HSGAGs to produceHSGAG oligosaccharides. MALDI (Matrix-Assisted Laser DesorptionIonization) mass spectrometry can be used for the identification andsemiquantitative measurement of substrates, enzymes, and end products inthe enzymatic reaction. The capillary electrophoresis techniqueseparates the products to resolve even small differences amongst theproducts and is applied in combination with mass spectrometry toquantitate the products produced. Capillary electrophoresis may evenresolve the difference between a disaccharide and its semicarbazonederivative. Detailed methods for sequencing polysaccharides and otherpolymers are disclosed in co-pending U.S. patent applications Ser. Nos.09/557,997 and 09/558,137, both filed on Apr. 24, 2000 and having commoninventorship. The entire contents of both applications are herebyincorporated by reference.

Briefly, the method is performed by enzymatic digestion, followed bymass spectrometry and capillary electrophoresis. The enzymatic assayscan be performed in a variety of manners, as long as the assays areperformed identically on the HSGAG, so that the results may be compared.In the example described in the Rhomberg reference, enzymatic reactionsare performed by adding 1 mL of enzyme solution to 5 mL of substratesolution. The digestion is then carried out at room temperature (22°C.), and the reaction is stopped at various time points by removing 0.5mL of the reaction mixture and adding it to 4.5 mL of a MALDI matrixsolution, such as caffeic acid (approximately 12 mg/mL) and 70%acetonitrile/water. The reaction mixture is then subjected to MALDI massspectrometry. The MALDI surface is prepared by the method of Xiang andBeavis (Xiang and Beavis (1994) Rapid. Commun. Mass. Spectrom. 8,199-204). A two-fold lower access of basic peptide (Arg/Gly)₁₅ ispremixed with matrix before being added to the oligosaccharide solution.A 1 mL aliquot of sample/matrix mixture containing 1-3 picomoles ofoligosaccharide is deposited on the surface. After crystallizationoccurs (typically within 60 seconds), excess liquid is rinsed off withwater. MALDI mass spectrometry spectra is then acquired in the linearmode by using a PerSeptive Biosystems (Framingham, Mass.) Voyager Elitereflectron time-of-flight instrument fitted with a 337 nanometernitrogen laser. Delayed extraction is used to increase resolution (22kV, grid at 93%, guidewire at 0.15%, pulse delay 150 ns, low mass gateat 1,000, 128 shots averaged). Mass spectra may be calibrated externallyby using the signals for proteinated (Arg/Gly)₁₅ and its complex withthe oligosaccharide.

Capillary electrophoresis may then be performed on aHewlett-Packard^(3D) CE unit by using uncoated fused silica capillaries(internal diameter 75 micrometers, outer diameter 363 micrometers,1_(det) 72.1 cm, and 1_(tot) 85 cm). Analytes are monitored by using UVdetection at 230 nm and an extended light path cell (Hewlett-Packard).The electrolyte is a solution of 10 mL dextran sulfate and 50 millimolarTris/phosphoric acid (pH2.5). Dextran sulfate is used to suppressnonspecific interactions of the heparin oligosaccharides with a silicawall. Separations are carried out at 30 kV with the anode at thedetector side (reversed polarity). A mixture of a1/5-naphtalenedisulfonic acid and 2-naphtalenesulfonic acid (10micromolar each) is used as an internal standard.

Other methods for assessing the product profile may also be utilized.For instance, other methods include methods which rely on parameterssuch as viscosity (Jandik, K. A., Gu, K. and Linhardt, R. J., (1994),Glycobiology, 4:284-296) or total UV absorbance (Ernst, S. et al.,(1996), Biochem. J, 315:589-597) or mass spectrometry or capillaryelectrophoresis alone.

The 2-O sulfatase molecules of the invention are also useful as toolsfor sequencing HSGAGs. Detailed methods for sequencing polysaccharidesand other polymers are disclosed in co-pending U.S. patent applicationsSer. Nos. 09/557,997 and 09/558,137, both filed on Apr. 24, 2000 andhaving common inventorship. These methods utilize tools such asheparinases in the sequencing process. The 2-O sulfatase of theinvention is useful as such a tool.

2-O sulfatase as well as the combinations of 2-O sulfatase with otherenzymes can, therefore, be used in any method of analyzing HSGAGs. Inaddition, these enzymes as described can be used to determine thepresence of a particular glycosaminoglycan in a sample or thecomposition of a glycosaminoglycans in a sample. A “sample”, as usedherein, refers to any sample that may contain a GAG.

One of ordinary skill in the art, in light of the present disclosure, isenabled to produce substantially pure preparations of HSGAG and/or GAGfragment compositions utilizing the 2-O sulfatase molecules alone or inconjunction with other enzymes. The GAG fragment preparations areprepared from HSGAG sources. A “HSGAG source” as used herein refers toheparin-like/heparan sulfate-like glycosaminoglycan composition whichcan be manipulated to produce GAG fragments using standard technology,including enzymatic degradation etc. As described above, HSGAGs includebut are not limited to isolated heparin, chemically modified heparin,biotechnology prepared heparin, synthetic heparin, heparan sulfate, andLMWH. Thus HSGAGs can be isolated from natural sources, prepared bydirect synthesis, mutagenesis, etc.

The 2-O sulfatase is, in some embodiments, immobilized on a support. The2-O sulfatase may be immobilized to any type of support but if thesupport is to be used in vivo or ex vivo it is desired that the supportis sterile and biocompatible. A biocompatible support is one which wouldnot cause an immune or other type of damaging reaction when used in asubject. The 2-O sulfatase may be immobilized by any method known in theart. Many methods are known for immobilizing proteins to supports. A“solid support” as used herein refers to any solid material to which apolypeptide can be immobilized.

Solid supports, for example, include but are not limited to membranes,e.g., natural and modified celluloses such as nitrocellulose or nylon,Sepharose, Agarose, glass, polystyrene, polypropylene, polyethylene,dextran, amylases, polyacrylamides, polyvinylidene difluoride, otheragaroses, and magnetite, including magnetic beads. The carrier can betotally insoluble or partially soluble and may have any possiblestructural configuration. Thus, the support may be spherical, as in abead, or cylindrical, as in the inside surface of a test tube ormicroplate well, or the external surface of a rod. Alternatively, thesurface may be flat such as a sheet, test strip, bottom surface of amicroplate well, etc.

The 2-O sulfatase of the invention may also be used to remove activeGAGs from a GAG containing fluid. A GAG containing fluid is contactedwith the 2-O sulfatase of the invention to degrade the GAG. The methodis particularly useful for the ex vivo removal of GAGs from blood. Inone embodiment of the invention the 2-O sulfatase is immobilized on asolid support as is conventional in the art. The solid supportcontaining the immobilized 2-O sulfatase may be used in extracorporealmedical devices (e.g. hemodialyzer, pump-oxygenator) for systemicheparinization to prevent the blood in the device from clotting. Thesupport membrane containing immobilized 2-O sulfatase is positioned atthe end of the device to neutralize the GAG before the blood is returnedto the body.

2-O sulfatase and the resulting GAG fragments also have many therapeuticutilities. A “therapeutic GAG fragment” as used herein refers to amolecule or molecules which are degraded GAGs or pieces or fragmentsthereof that have been degraded through the use of the 2-O sulfatasepossibly along with other GAG-degrading enzymes, (e.g. native and/ormodified heparinases). Such compounds may be generated using 2-Osulfatase to produce therapeutic fragments or they may be synthesized denovo. Putative GAG fragments can be tested for therapeutic activityusing any of the assays described herein or known in the art. Thus thetherapeutic GAG fragment may be a synthetic GAG fragment generated basedon the sequence of the GAG fragment identified when the tumor iscontacted with 2-O sulfatase, or having minor variations which do notinterfere with the activity of the compound. Alternatively thetherapeutic GAG fragment may be an isolated GAG fragment produced whenthe tumor is contacted with 2-O sulfatase.

The 2-O sulfatase and/or GAG fragments can be used for the treatment ofany type of condition in which GAG fragment therapy has been identifiedas a useful therapy, such as preventing coagulation, inhibitingangiogenesis, preventing neovascularization, inhibiting proliferation,regulating apoptosis, etc. The methods of the invention also enable oneof skill in the art to prepare or identify an appropriate composition ofGAG fragments, depending on the subject and the disorder being treated.These compositions of GAG fragments may be used alone or in combinationwith the 2-O sulfatase and/or other enzymes. Likewise 2-O sulfataseand/or other enzymes may also be used to produce GAG fragments in vivo.

The invention is useful for treating and/or preventing anydisease/condition in a subject whereby glycosaminoglycans have beenfound to be important in the development and/or progress of the disease.The terms “treat” and “treating” as used herein refers to reversing orblocking the progression of the disease in the subject. Treating adisease also includes exacting a desired improvement in the disease orsymptoms of the disease. For example to treat a subject with tumor cellproliferation refers to inhibiting completely or partially theproliferation or metastasis of a cancer or tumor cell, as well asinhibiting or preventing any increase in the proliferation or metastasisof a cancer or tumor cell.

A “subject having a disease” is a subject that can be diagnosed ashaving the disease, e.g., a person having cancer is identified by thepresence of cancerous cells. A “subject at risk of having a disease” asused herein is a subject who has a high probability of developing thedisease. These subjects include, for instance, subjects having a geneticabnormality, the presence of which has been demonstrated to have acorrelative relation to a higher likelihood of developing the disease.For diseases brought about by exposure to disease causing agents,subjects at risk are those who are exposed to the disease causing agentssuch as tobacco, asbestos, chemical toxins, viruses, parasites, etc. Asubject at risk also includes those who have previously been treated forthe disease and have the possibility of having a recurrence of thedisease. When a subject at risk of developing a disease is treated witha 2-O sulfatase, a cocktail of 2-O sulfatase along with otherGAG—degrading enzymes (e.g. heparinase and Δ4, 5 glycuronidase) ordegradation products thereof the subject is able to prevent theoccurrence of the disease or reduce the possibility of developing thedisease.

The compositions of the invention, therefore, can be used for thetreatment of any type of condition in which GAG fragment therapy hasbeen identified as a useful therapy. Thus, the invention is useful in avariety of in vitro, in vivo and ex vivo methods in which therapies areuseful. For instance, GAG fragments can also be useful for treating orpreventing cancer, atherosclerosis, neurodegenerative disease (e.g.Alzheimer's), microbial infection, psoriasis, etc. GAG fragments canalso be useful in tissue repair. The GAG fragment compositions may alsobe used in in vitro assays, such as a quality control sample.

Each of these disorders mentioned herein is well-known in the art and isdescribed, for instance, in Harrison 's Principles of Internal Medicine(McGraw Hill, Inc., New York), which is incorporated by reference.

In one embodiment the preparations of the invention are used forinhibiting angiogenesis. An effective amount for inhibiting angiogenesisof the GAG fragment preparation is administered to a subject in need oftreatment thereof. Angiogenesis as used herein is the inappropriateformation of new blood vessels. “Angiogenesis” often occurs in tumorswhen endothelial cells secrete a group of growth factors that aremitogenic for endothelium causing the elongation and proliferation ofendothelial cells which results in a generation of new blood vessels.Several of the angiogenic mitogens are heparin binding peptides whichare related to endothelial cell growth factors. The inhibition ofangiogenesis can cause tumor regression in animal models, suggesting ause as a therapeutic anticancer agent. An effective amount forinhibiting angiogenesis is an amount of GAG fragment preparation whichis sufficient to diminish the number of blood vessels growing into atumor. This amount can be assessed in an animal model of tumors andangiogenesis, many of which are known in the art.

Thus, the 2-O sulfatase molecules are useful for treating or preventingdisorders associated with coagulation. A “disease associated withcoagulation” as used herein refers to a condition characterized by aninterruption in the blood supply to a tissue due to a blockage of theblood vessel responsible for supplying blood to the tissue such as isseen for myocardial or cerebral infarction. A cerebral ischemic attackor cerebral ischemia is a form of ischemic condition in which the bloodsupply to the brain is blocked. This interruption in the blood supply tothe brain may result from a variety of causes, including an intrinsicblockage or occlusion of the blood vessel itself, a remotely originatedsource of occlusion, decreased perfusion pressure or increased bloodviscosity resulting in inadequate cerebral blood flow, or a rupturedblood vessel in the subarachnoid space or intracerebral tissue.

A “disease associated with coagulation” as used herein also is intendedto encompass atherosclerosis. Atherosclerosisis a disease of thearteries whereby blood flow can be reduced due to the development ofatheromatous plaques along the interior walls of the arteries. Theseplaques begin by the initial deposition of cholesterol crystals whichgrow larger with time. In addition to the cholesterol deposition,plaques also grow due to the proliferation of the surrounding cells. Intime, the artery may become completely occluded due to this plaquegrowth.

The 2-O sulfatase or the GAG fragments generated therewith may be usedalone or in combination with a therapeutic agent for treating a diseaseassociated with coagulation. Examples of therapeutics useful in thetreatment of diseases associated with coagulation includeanticoagulation agents, antiplatelet agents, and thrombolytic agents.

Anticoagulation agents prevent the coagulation of blood components andthus prevent clot formation. Anticoagulants include, but are not limitedto, heparin, warfarin, coumadin, dicumarol, phenprocoumon,acenocoumarol, ethyl biscoumacetate, and indandione derivatives.

Antiplatelet agents inhibit platelet aggregation and are often used toprevent thromboembolic stroke in patients who have experienced atransient ischemic attack or stroke. Antiplatelet agents include, butare not limited to, aspirin, thienopyridine derivatives such asticlopodine and clopidogrel, dipyridamole and sulfinpyrazone, as well asRGD mimetics and also antithrombin agents such as, but not limited to,hirudin.

Thrombolytic agents lyse clots which cause the thromboembolic stroke.Thrombolytic agents have been used in the treatment of acute venousthromboembolism and pulmonary emboli and are well known in the art (e.g.see Hennekens et al, J Am Coll Cardiol; v. 25 (7 supp), p. 18S-22S(1995); Holmes, et al, J Am Coll Cardiol; v.25 (7 suppl), p.10S-17S(1995)). Thrombolytic agents include, but are not limited to,plasminogen, a₂-antiplasmin, streptokinase, antistreplase, tissueplasminogen activator (tPA), and urokinase. “tPA” as used hereinincludes native tPA and recombinant tPA, as well as modified forms oftPA that retain the enzymatic or fibrinolytic activities of native tPA.The enzymatic activity of tPA can be measured by assessing the abilityof the molecule to convert plasminogen to plasmin. The fibrinolyticactivity of tPA may be determined by any in vitro clot lysis activityknown in the art, such as the purified clot lysis assay described byCarlson, et. al., Anal. Biochem. 168, 428-435 (1988) and its modifiedform described by Bennett, W. F. et al., 1991, J. Biol. Chem.266(8):5191-5201, the entire contents of which are hereby incorporatedby reference.

The compositions as described herein can also be used to prevent ortreat “neurodegenerative disease” is defined herein as a disease inwhich progressive loss of neurons occurs either in the peripheralnervous system or in the central nervous system. Examples ofneurodegenerative disorders include familial and sporadic amyotrophiclateral sclerosis (FALS and ALS, respectively), familial and sporadicParkinson's disease, Huntington's disease, familial and sporadicAlzheimer's disease, multiple sclerosis, olivopontocerebellar atrophy,multiple system atrophy, progressive supranuclear palsy, diffuse Lewybody disease, corticodentatonigral degeneration, progressive familialmyoclonic epilepsy, strionigral degeneration, torsion dystonia, familialtremor, Down's Syndrome, Gilles de la Tourette syndrome,Hallervorden-Spatz disease, diabetic peripheral neuropathy, dementiapugilistica, AIDS dementia, age related dementia, age associated memoryimpairment, amyloidosis-related neurodegenerative diseases such as thosecaused by the prion protein (PrP) which is associated with transmissiblespongiform encephalopathy (Creutzfeldt-Jakob disease,Gerstmann-Straussler-Scheinker syndrome, scrapie, bovine spongiformencephalopathy and kuru), and those caused by excess cystatin Caccumulation (hereditary cystatin C angiopathy), traumatic brain injury(e.g., surgery-related brain injury), cerebral edema, peripheral nervedamage, spinal cord injury, Wemicke-Korsakoff's related dementia(alcohol induced dementia), and presenile dementia. The foregoingexamples are not meant to be comprehensive but serve merely as anillustration of the term “neurodegenerative disease”.

The invention also provides treatment or prevention of aneurodegenerative disease by the administration of the 2-O sulfataseand/or GAG fragment compositions described herein possibly inconjunction with other therapeutic agents for the particular conditionbeing treated. The administration the other therapeutics may beperformed concomitantly, sequentially or at different time points.

For example, when treating Alzheimer's Disease, the therapeutic agentswhich can be combined with the compositions of the invention include,but are not limited to, estrogen, vitamin E (alpha-tocopherol), Tacrine(tetrahydroacridinamine), selegiline (deprenyl), and Aracept(donepezil). One of ordinary skill in the art will be familiar withadditional therapeutic agents useful for the treatment ofneurodegenerative diseases.

Critically, HSGAGs (along with collagen) are key components of the cellsurface-extracellular matrix (ECM) interface. While collagen-likeproteins provide the necessary extracellular scaffold for cells toattach and form tissues, the complex polysaccharides fill the spacecreated by the scaffold and act as a molecular sponge by specificallybinding and regulating the biological activities of numerous signalingmolecules like growth factors, cytokines etc. Therefore, thecompositions provided herein can also be used in methods of repairingtissues.

In addition, as it had been found that viruses and parasites utilizeglycosaminoglycans such as heparan sulfate as receptors to infect targetcells (Liu, J., and Thorp, S. C. (2002) Med Res Rev 22(1), 1-25), thecompositions of the invention may also be used to treat or preventmicrobial infections. The compositions of the invention can also beadministered in combination with other antiviral agents or antiparasiticagents.

Antiviral agents are compounds which prevent infection of cells byviruses or replication of the virus within the cell. There are severalstages within the process of viral infection which can be blocked orinhibited by antiviral agents. These stages include, attachment of thevirus to the host cell (immunoglobulin or binding peptides), uncoatingof the virus (e.g., amantadine), synthesis or translation of viral mRNA(e.g., interferon), replication of viral RNA or DNA (e.g., nucleosideanalogues), maturation of new virus proteins (e.g., proteaseinhibitors), and budding and release of the virus.

Examples of antiviral agents known in the art are nucleotide analogueswhich include, but are not limited to, acyclovir (used for the treatmentof herpes simplex virus and varicella-zoster virus), gancyclovir (usefulfor the treatment of cytomegalovirus), idoxuridine, ribavirin (usefulfor the treatment of respiratory syncitial virus), dideoxyinosine,dideoxycytidine, and zidovudine (azidothymidine).

It has also been recently been recognized that cells synthesize distinctHSGAG sequences and decorate themselves with these sequences, using theextraordinary information content present in the sequences to bindspecifically to many signaling molecules and thereby regulate variousbiological processes. The processes include apoptosis (Ishikawa, Y., andKitamura, M. (1999) Kidney Int 56(3), 954-63, Kapila, Y. L., Wang, S.,Dazin, P., Tafolla, E., and Mass, M. J. (2002) J Biol Chem 277(10),8482-91). Regulation of apoptosis with the compositions of the inventioncan prove important to a variety of diseases whereby an increase ordecrease in cell death is warranted. Apoptosis is known to play a rolein numerous physiologic and pathologic events such as embryogenesis andmetamorphosis, hormone-dependent involution in the adult, cell death intumors, atrophy of some organs and tissues, etc.

As the compositions of the invention are useful for the same purposes asheparinases and the degradation products of heparinases (HSGAGfragments), they are also useful for treating and preventing cancer cellproliferation and metastasis. Thus, according to another aspect of theinvention, there is provided methods for treating subjects having or atrisk of having cancer. The cancer may be a malignant or non-malignantcancer. Cancers or tumors include but are not limited to biliary tractcancer; brain cancer; breast cancer; cervical cancer; choriocarcinoma;colon cancer; endometrial cancer; esophageal cancer; gastric cancer;intraepithelial neoplasms; lymphomas; liver cancer; lung cancer (e.g.small cell and non-small cell); melanoma; neuroblastomas; oral cancer;ovarian cancer; pancreas cancer; prostate cancer; rectal cancer;sarcomas; skin cancer; testicular cancer; thyroid cancer; and renalcancer, as well as other carcinomas and sarcomas.

The invention also encompasses screening assays for identifyingtherapeutic GAG fragments for the treatment of a tumor and forpreventing metastasis. The assays are accomplished by treating a tumoror isolated tumor cells with 2-O sulfatase and/or other native ormodified heparinases and isolating the resultant GAG fragments.Surprisingly, these GAG fragments have therapeutic activity in theprevention of tumor cell proliferation and metastasis. Thus theinvention encompasses individualized therapies, in which a tumor orportion of a tumor is isolated from a subject and used to prepare thetherapeutic GAG fragments. These therapeutic fragments can bere-administered to the subject to protect the subject from further tumorcell proliferation or metastasis or from the initiation of metastasis ifthe tumor is not yet metastatic. Alternatively the fragments can be usedin a different subject having the same type or tumor or a different typeof tumor.

The invasion and metastasis of cancer is a complex process whichinvolves changes in cell adhesion properties which allow a transformedcell to invade and migrate through the extracellular matrix (ECM) andacquire anchorage-independent growth properties (Liotta, L. A., et al.,Cell 64:327-336, 1991). Some of these changes occur at focal adhesions,which are cell/ECM contact points containing membrane-associated,cytoskeletal, and intracellular signaling molecules. Metastatic diseaseoccurs when the disseminated foci of tumor cells seed a tissue whichsupports their growth and propagation, and this secondary spread oftumor cells is responsible for the morbidity and mortality associatedwith the majority of cancers. Thus the term “metastasis” as used hereinrefers to the invasion and migration of tumor cells away from theprimary tumor site.

The barrier for the tumor cells may be an artificial barrier in vitro ora natural barrier in vivo. In vitro barriers include but are not limitedto extracellular matrix coated membranes, such as Matrigel. Thus the 2-Osulfatase compositions or degradation products thereof can be tested fortheir ability to inhibit tumor cell invasion in a Matrigel invasionassay system as described in detail by Parish, C. R., et al., “ABasement-Membrane Permeability Assay which Correlates with theMetastatic Potential of Tumour Cells,” Int. J. Cancer, 1992, 52:378-383.Matrigel is a reconstituted basement membrane containing type IVcollagen, laminin, heparan sulfate proteoglycans such as perlecan, whichbind to and localize bFGF, vitronectin as well as transforming growthfactor-β (TGF-β), urokinase-type plasminogen activator (uPA), tissueplasminogen activator (tPA), and the serpin known as plasminogenactivator inhibitor type 1 (PAI-1). Other in vitro and in vivo assaysfor metastasis have been described in the prior art, see, e.g., U.S.Pat. No. 5,935,850, issued on Aug. 10, 1999, which is incorporated byreference. An in vivo barrier refers to a cellular barrier present inthe body of a subject.

Effective amounts of the 2-O sulfatase, functional variants thereof ortherapeutic GAGs of the invention are administered to subjects in needof such treatment. Effective amounts are those amounts which will resultin a desired improvement in the condition or symptoms of the condition,e.g., for cancer this is a reduction in cellular proliferation ormetastasis, without causing other medically unacceptable side effects.Such amounts can be determined with no more than routineexperimentation. It is believed that doses ranging from 1nanogram/kilogram to 100 milligrams/kilogram, depending upon the mode ofadministration, will be effective. The absolute amount will depend upona variety of factors (including whether the administration is inconjunction with other methods of treatment, the number of doses andindividual patient parameters including age, physical condition, sizeand weight) and can be determined with routine experimentation. It ispreferred generally that a maximum dose be used, that is, the highestsafe dose according to sound medical judgment. The mode ofadministration may be any medically acceptable mode including oral,subcutaneous, intravenous, etc.

In general, when administered for therapeutic purposes, the formulationsof the invention are applied in pharmaceutically acceptable solutions.Such preparations may routinely contain pharmaceutically acceptableconcentrations of salt, buffering agents, preservatives, compatiblecarriers, adjuvants, and optionally other therapeutic ingredients.

The compositions of the invention may be administered per se (neat) orin the form of a pharmaceutically acceptable salt. When used in medicinethe salts should be pharmaceutically acceptable, butnon-pharmaceutically acceptable salts may conveniently be used toprepare pharmaceutically acceptable salts thereof and are not excludedfrom the scope of the invention. Such pharmacologically andpharmaceutically acceptable salts include, but are not limited to, thoseprepared from the following acids: hydrochloric, hydrobromic, sulphuric,nitric, phosphoric, maleic, acetic, salicylic, p-toluene sulphonic,tartaric, citric, methane sulphonic, formic, malonic, succinic,naphthalene-2-sulphonic, and benzene sulphonic. Also, pharmaceuticallyacceptable salts can be prepared as alkaline metal or alkaline earthsalts, such as sodium, potassium or calcium salts of the carboxylic acidgroup.

Suitable buffering agents include: acetic acid and a salt (1-2% W/V);citric acid and a salt (1-3% W/V); boric acid and a salt (0.5-2.5% W/V);and phosphoric acid and a salt (0.8-2% W/V). Suitable preservativesinclude benzalkonium chloride (0.003-0.03% W/V); chlorobutanol (0.3-0.9%W/V); parabens (0.01-0.25% W/V) and thimerosal (0.004-0.02% W/V).

The present invention provides pharmaceutical compositions, for medicaluse, which comprise 2-O sulfatase, functional variants thereof ortherapeutic GAG fragments together with one or more pharmaceuticallyacceptable carriers and optionally other therapeutic ingredients. Theterm “pharmaceutically-acceptable carrier” as used herein, and describedmore fully below, means one or more compatible solid or liquid filler,dilutants or encapsulating substances which are suitable foradministration to a human or other animal. In the present invention, theterm “carrier” denotes an organic or inorganic ingredient, natural orsynthetic, with which the active ingredient is combined to facilitatethe application. The components of the pharmaceutical compositions alsoare capable of being commingled with the 2-O sulfatase of the presentinvention or other compositions, and with each other, in a manner suchthat there is no interaction which would substantially impair thedesired pharmaceutical efficiency.

A variety of administration routes are available. The particular modeselected will depend, of course, upon the particular active agentselected, the particular condition being treated and the dosage requiredfor therapeutic efficacy. The methods of this invention, generallyspeaking, may be practiced using any mode of administration that ismedically acceptable, meaning any mode that produces effective levels ofan immune response without causing clinically unacceptable adverseeffects. A preferred mode of administration is a parenteral route. Theterm “parenteral” includes subcutaneous injections, intravenous,intramuscular, intraperitoneal, intra sternal injection or infusiontechniques. Other modes of administration include oral, mucosal, rectal,vaginal, sublingual, intranasal, intratracheal, inhalation, ocular,transdermal, etc.

For oral administration, the compounds can be formulated readily bycombining the active compound(s) with pharmaceutically acceptablecarriers well known in the art. Such carriers enable the compounds ofthe invention to be formulated as tablets, pills, dragees, capsules,liquids, gels, syrups, slurries, suspensions and the like, for oralingestion by a subject to be treated. Pharmaceutical preparations fororal use can be obtained as solid excipient, optionally grinding aresulting mixture, and processing the mixture of granules, after addingsuitable auxiliaries, if desired, to obtain tablets or dragee cores.Suitable excipients are, in particular, fillers such as sugars,including lactose, sucrose, mannitol, or sorbitol; cellulosepreparations such as, for example, maize starch, wheat starch, ricestarch, potato starch, gelatin, gum tragacanth, methyl cellulose,hydroxypropylmethyl-cellulose, sodium carboxymethylcellulose, and/orpolyvinylpyrrolidone (PVP). If desired, disintegrating agents may beadded, such as the cross-linked polyvinyl pyrrolidone, agar, or alginicacid or a salt thereof such as sodium alginate. Optionally the oralformulations may also be formulated in saline or buffers forneutralizing internal acid conditions or may be administered without anycarriers.

Dragee cores are provided with suitable coatings. For this purpose,concentrated sugar solutions may be used, which may optionally containgum arabic, talc, polyvinyl pyrrolidone, carbopol gel, polyethyleneglycol, and/or titanium dioxide, lacquer solutions, and suitable organicsolvents or solvent mixtures. Dyestuffs or pigments may be added to thetablets or dragee coatings for identification or to characterizedifferent combinations of active compound doses.

Pharmaceutical preparations which can be used orally include push-fitcapsules made of gelatin, as well as soft, sealed capsules made ofgelatin and a plasticizer, such as glycerol or sorbitol. The push-fitcapsules can contain the active ingredients in admixture with fillersuch as lactose, binders such as starches, and/or lubricants such astalc or magnesium stearate and, optionally, stabilizers. In softcapsules, the active compounds may be dissolved or suspended in suitableliquids, such as fatty oils, liquid paraffin, or liquid polyethyleneglycols. In addition, stabilizers may be added. Microspheres formulatedfor oral administration may also be used. Such microspheres have beenwell defined in the art. All formulations for oral administration shouldbe in dosages suitable for such administration.

For buccal administration, the compositions may take the form of tabletsor lozenges formulated in conventional manner.

For administration by inhalation, the compounds for use according to thepresent invention may be conveniently delivered in the form of anaerosol spray presentation from pressurized packs or a nebulizer, withthe use of a suitable propellant, e.g., dichlorodifluoromethane,trichlorofluoromethane, dichlorotetrafluoroethane, carbon dioxide orother suitable gas. In the case of a pressurized aerosol the dosage unitmay be determined by providing a valve to deliver a metered amount.Capsules and cartridges of e.g. gelatin for use in an inhaler orinsufflator may be formulated containing a powder mix of the compoundand a suitable powder base such as lactose or starch.

The compounds, when it is desirable to deliver them systemically, may beformulated for parenteral administration by injection, e.g., by bolusinjection or continuous infusion. Formulations for injection may bepresented in unit dosage form, e.g., in ampoules or in multi-dosecontainers, with an added preservative. The compositions may take suchforms as suspensions, solutions or emulsions in oily or aqueousvehicles, and may contain formulatory agents such as suspending,stabilizing and/or dispersing agents.

Pharmaceutical formulations for parenteral administration includeaqueous solutions of the active compounds in water-soluble form.Additionally, suspensions of the active compounds may be prepared asappropriate oily injection suspensions. Suitable lipophilic solvents orvehicles include fatty oils such as sesame oil, or synthetic fatty acidesters, such as ethyl oleate or triglycerides, or liposomes. Aqueousinjection suspensions may contain substances which increase theviscosity of the suspension, such as sodium carboxymethyl cellulose,sorbitol, or dextran. Optionally, the suspension may also containsuitable stabilizers or agents which increase the solubility of thecompounds to allow for the preparation of highly concentrated solutions.

Alternatively, the active compounds may be in powder form forconstitution with a suitable vehicle, e.g., sterile pyrogen-free water,before use.

The compounds may also be formulated in rectal or vaginal compositionssuch as suppositories or retention enemas, e.g., containing conventionalsuppository bases such as cocoa butter or other glycerides.

In addition to the formulations described previously, the compounds mayalso be formulated as a depot preparation. Such long acting formulationsmay be formulated with suitable polymeric or hydrophobic materials (forexample as an emulsion in an acceptable oil) or ion exchange resins, oras sparingly soluble derivatives, for example, as a sparingly solublesalt.

The pharmaceutical compositions also may comprise suitable solid or gelphase carriers or excipients. Examples of such carriers or excipientsinclude but are not limited to calcium carbonate, calcium phosphate,various sugars, starches, cellulose derivatives, gelatin, and polymerssuch as polyethylene glycols.

Suitable liquid or solid pharmaceutical preparation forms are, forexample, aqueous or saline solutions for inhalation, microencapsulated,encochleated, coated onto microscopic gold particles, contained inliposomes, nebulized, aerosols, pellets for implantation into the skin,or dried onto a sharp object to be scratched into the skin. Thepharmaceutical compositions also include granules, powders, tablets,coated tablets, (micro)capsules, suppositories, syrups, emulsions,suspensions, creams, drops or preparations with protracted release ofactive compounds, in whose preparation excipients and additives and/orauxiliaries such as disintegrants, binders, coating agents, swellingagents, lubricants, flavorings, sweeteners or solubilizers arecustomarily used as described above. The pharmaceutical compositions aresuitable for use in a variety of drug delivery systems. For a briefreview of methods for drug delivery, see Langer, Science 249:1527-1533,1990, which is incorporated herein by reference.

The compositions may conveniently be presented in unit dosage form andmay be prepared by any of the methods well known in the art of pharmacy.All methods include the step of bringing the active 2-O sulfatase intoassociation with a carrier which constitutes one or more accessoryingredients. In general, the compositions are prepared by uniformly andintimately bringing the polymer into association with a liquid carrier,a finely divided solid carrier, or both, and then, if necessary, shapingthe product. The compositions may be stored lyophilized.

Other delivery systems can include time-release, delayed release orsustained release delivery systems. Such systems can avoid repeatedadministrations of the heparinases of the invention, increasingconvenience to the subject and the physician. Many types of releasedelivery systems are available and known to those of ordinary skill inthe art. They include polymer based systems such as polylactic andpolyglycolic acid, polyanhydrides and polycaprolactone; nonpolymersystems that are lipids including sterols such as cholesterol,cholesterol esters and fatty acids or neutral fats such as mono-, di andtriglycerides; hydrogel release systems; silastic systems; peptide basedsystems; wax coatings, compressed tablets using conventional binders andexcipients, partially fused implants and the like. Specific examplesinclude, but are not limited to: (a) erosional systems in which thepolysaccharide is contained in a form within a matrix, found in U.S.Pat. No. 4,452,775 (Kent); U.S. Pat. No. 4,667,014 (Nestor et al.); andU.S. Pat. Nos. 4,748,034 and 5,239,660 (Leonard) and (b) diffusionalsystems in which an active component permeates at a controlled ratethrough a polymer, found in U.S. Pat. No. 3,832,253 (Higuchi et al.) andU.S. Pat. No. 3,854,480 (Zaffaroni). In addition, a pump-based hardwaredelivery system can be used, some of which are adapted for implantation.

A subject is any human or non-human vertebrate, e.g., dog, cat, horse,cow, pig.

When administered to a patient undergoing cancer treatment, the 2-Osulfatase or therapeutic GAG compounds may be administered in cocktailscontaining other anti-cancer agents. The compounds may also beadministered in cocktails containing agents that treat the side-effectsof radiation therapy, such as anti-emetics, radiation protectants, etc.

Anti-cancer drugs that can be co-administered with the compounds of theinvention include, but are not limited to Acivicin; Aclarubicin;Acodazole Hydrochloride; Acronine; Adriamycin; Adozelesin; Aldesleukin;Altretamine; Ambomycin; Ametantrone Acetate; Aminoglutethimide;Amsacrine; Anastrozole; Anthramycin; Asparaginase; Asperlin;Azacitidine; Azetepa; Azotomycin; Batimastat; Benzodepa; Bicalutamide;Bisantrene Hydrochloride; Bisnafide Dimesylate; Bizelesin; BleomycinSulfate; Brequinar Sodium; Bropirimine; Busulfan; Cactinomycin;Calusterone; Caracemide; Carbetimer; Carboplatin; Carmustine; CarubicinHydrochloride; Carzelesin; Cedefingol; Chlorambucil; Cirolemycin;Cisplatin; Cladribine; Crisnatol Mesylate; Cyclophosphamide; Cytarabine;Dacarbazine; Dactinomycin; Daunorubicin Hydrochloride; Decitabine;Dexormaplatin; Dezaguanine; Dezaguanine Mesylate; Diaziquone; Docetaxel;Doxorubicin; Doxorubicin Hydrochloride; Droloxifene; DroloxifeneCitrate; Dromostanolone Propionate; Duazomycin; Edatrexate; EflomithineHydrochloride; Elsamitrucin; Enloplatin; Enpromate; Epipropidine;Epirubicin Hydrochloride; Erbulozole; Esorubicin Hydrochloride;Estramustine; Estramustine Phosphate Sodium; Etanidazole; Etoposide;Etoposide Phosphate; Etoprine; Fadrozole Hydrochloride; Fazarabine;Fenretinide; Floxuridine; Fludarabine Phosphate; Fluorouracil;Flurocitabine; Fosquidone; Fostriecin Sodium; Gemcitabine; GemcitabineHydrochloride; Hydroxyurea; Idarubicin Hydrochloride; Ifosfamide;Ilmofosine; Interferon Alfa-2a; Interferon Alfa-2b; Interferon Alfa-n1;Interferon Alfa-n3; Interferon Beta-I a; Interferon Gamma-I b;Iproplatin; Irinotecan Hydrochloride; Lanreotide Acetate; Letrozole;Leuprolide Acetate; Liarozole Hydrochloride; Lometrexol Sodium;Lomustine; Losoxantrone Hydrochloride; Masoprocol; Maytansine;Mechlorethamine Hydrochloride; Megestrol Acetate; Melengestrol Acetate;Melphalan; Menogaril; Mercaptopurine; Methotrexate; Methotrexate Sodium;Metoprine; Meturedepa; Mitindomide; Mitocarcin; Mitocromin; Mitogillin;Mitomalcin; Mitomycin; Mitosper; Mitotane; Mitoxantrone Hydrochloride;Mycophenolic Acid; Nocodazole; Nogalamycin; Ormaplatin; Oxisuran;Paclitaxel; Pegaspargase; Peliomycin; Pentamustine; Peplomycin Sulfate;Perfosfamide; Pipobroman; Piposulfan; Piroxantrone Hydrochloride;Plicamycin; Plomestane; Porfimer Sodium; Porfiromycin; Prednimustine;Procarbazine Hydrochloride; Puromycin; Puromycin Hydrochloride;Pyrazofurin; Riboprine; Rogletimide; Safingol; Safingol Hydrochloride;Semustine; Simtrazene; Sparfosate Sodium; Sparsomycin; SpirogermaniumHydrochloride; Spiromustine; Spiroplatin; Streptonigrin; Streptozocin;Sulofenur; Talisomycin; Tecogalan Sodium; Tegafur; TeloxantroneHydrochloride; Temoporfin; Teniposide; Teroxirone; Testolactone;Thiamiprine; Thioguanine; Thiotepa; Tiazofurin; Tirapazamine; TopotecanHydrochloride; Toremifene Citrate; Trestolone Acetate; TriciribinePhosphate; Trimetrexate; Trimetrexate Glucuronate; Triptorelin;Tubulozole Hydrochloride; Uracil Mustard; Uredepa; Vapreotide;Verteporfin; Vinblastine Sulfate; Vincristine Sulfate; Vindesine;Vindesine Sulfate; Vinepidine Sulfate; Vinglycinate Sulfate;Vinleurosine Sulfate; Vinorelbine Tartrate; Vinrosidine Sulfate;Vinzolidine Sulfate; Vorozole; Zeniplatin; Zinostatin; ZorubicinHydrochloride.

The 2-O sulfatase or therapeutic GAG compounds may also be linked to atargeting molecule. A targeting molecule is any molecule or compoundwhich is specific for a particular cell or tissue and which can be usedto direct the 2-O sulfatase or therapeutic GAG to the cell or tissue.Preferably the targeting molecule is a molecule which specificallyinteracts with a cancer cell or a tumor. For instance, the targetingmolecule may be a protein or other type of molecule that recognizes andspecifically interacts with a tumor antigen.

Tumor-antigens include Melan-A/MART-1, Dipeptidyl peptidase IV (DPPIV),adenosine deaminase-binding protein (ADAbp), cyclophilin b, Colorectalassociated antigen (CRC)-C017-1A/GA733, Carcinoembryonic Antigen (CEA)and its immunogenic epitopes CAP-1 and CAP-2, etv6, aml1, ProstateSpecific Antigen (PSA) and its immunogenic epitopes PSA-1, PSA-2, andPSA-3, prostate-specific membrane antigen (PSMA), T-cellreceptor/CD3-zeta chain, MAGE-family of tumor antigens (e.g., MAGE-A1,MAGE-A2, MAGE-A3, MAGE-A4, MAGE-A5, MAGE-A6, MAGE-A7, MAGE-A8, MAGE-A9,MAGE-A10, MAGE-A11, MAGE-A12, MAGE-Xp2 (MAGE-B2), MAGE-Xp3 (MAGE-B3),MAGE-Xp4 (MAGE-B4), MAGE-C1, MAGE-C2, MAGE-C3, MAGE-C4, MAGE-C5),GAGE-family of tumor antigens (e.g., GAGE-1, GAGE-2, GAGE-3, GAGE-4,GAGE-5, GAGE-6, GAGE-7, GAGE-8, GAGE-9), BAGE, RAGE, LAGE-1, NAG, GnT-V,MUM-1, CDK4, tyrosinase, p53, MUC family, HER2/neu, p21ras, RC1SI,α-fetoprotein, E-cadherin, α-catenin, β-catenin and γ-catenin, p120ctn,gp100^(Pmel117), PRAME, NY-ESO-1, brain glycogen phosphorylase, SSX-1,SSX-2 (HOM-MEL-40), SSX-1, SSX-4, SSX-5, SCP-1, CT-7, cdc27, adenomatouspolyposis coli protein (APC), fodrin, P1A, Connexin 37, Ig-idiotype,p15, gp75, GM2 and GD2 gangliosides, viral products such as humanpapilloma virus proteins, Smad family of tumor antigens, lmp-1,EBV-encoded nuclear antigen (EBNA)-1, and c-erbB-2.

The present invention is further illustrated by the following Examples,which in no way should be construed as further limiting. The entirecontents of all of the references (including literature references,issued patents, published patent applications, and co-pending patentapplications) cited throughout this application are hereby expresslyincorporated by reference.

EXAMPLES

Materials And Methods

Reagents—Heparin and chondroitin disaccaharides were purchased fromCalbiochem (La Jolla, Calif.). Unfractionated heparin was obtained fromCelsus Laboratories (Cincinatti, Ohio). The unsaturated heparintetrasaccharide ΔU_(2S)H_(NS,6S)I_(2S)H_(NS,6S) (T1) and decasaccharideΔU_(2S)H_(NS,6S)I_(2S)H_(NS,6S)I_(2S)H_(NS,6S)IH_(NAc,6S)GH_(NS,3S,6S)(AT-10) were generated by a partial heparinase digestion and purified asdescribed (Toida, T., Hileman, R. E., Smith, A. E., Vlahova, P.I., andLinhardt, R. J. (1996) J Biol Chem 271(50),32040-7). Materials for λZAPII genomic library construction, screening and phagemid excisionincluding bacteriophage host strain XLIBlue MRF and the helper-resistantstrain SOLR were obtained from Stratagene (La Jolla, Calif.) and usedaccording to the Manufacturer's instructions. Restriction endonucleasesand molecular cloning and PCR enzymes were purchased from New EnglandBiolabs (Beverly, Mass.). DNA oligonucleotide primers were synthesizedby Invitrogen/Life Technologies custom primer service (Carlisbad,Calif.). TOP10 chemically competent cells for PCR cloning and subdloningwere also obtained from Invitrogen. [³²P]dCTP radionuclides werepurchased from NEN (Boston, Mass.). Additional molecular cloningreagents were obtained from the manufacturers listed. Modified trypsin(sequencing grade) was purchased from Roche Molecular Biochemicals(Indianapolis, Ind.). Texas Red hydrazine was purchased from MolecularProbes (Eugene, Oreg.). All other reagents were from Sigma-Aldrich (St.Louis, Mo.) unless otherwise noted.Purification of the Flavobacterium heparinum 2-O sulfatase andsubsequent proteolysis—The 2-O sulfatase was purified from 20 literfermentation cultures. Briefly, the large-scale cultures were grown at25° C. for 48 hours. Cell lysates were obtained by a repeated passage ofa resuspended cell pellet through an Aminco French-pressure cell. Thehomogenate was clarified by centrifugation (37000×g). The 2-O sulfatasewas purified from this cell-free supernatant by employing fivechromatographic steps carried out in the following sequence:cation-exchange (CM-Sepaharose CL-6B)→hydroxyapatite (Bio-Gel HTP)→gelfiltration (Sephadex G-50)→taurine-Sepharose CL-4B→blue-Sepharose CL-6B.2-O sulfatase activity was measured at each chromatography step asdescribed (McLean, M. W., Bruce, J. S., Long, W. F., and Williamson, F.B. (1984) Eur J Biochem 145(3), 607-15). Fractions from 6 initialCM-sepharose chromatography were also assayed for heparinase,chondroitinase (AC and B) and Δ 4,5 glycuronidase activities as well asany co-eluting 6-O or N sulfatase activities. The highly purified 2-Osulfatase pool from the final blue-Sepharose chromatography step wasfree from any contaminating glycosaminoglycan degrading activity.Generation of 2-O sulfatase peptides and protein sequencing—Inpreparation for proteolysis, the purified flavobacterial sulfatase wasfirst desalted by reverse phase chromatography (RP-HPLC) on a 150 mm×4.6mm C4 column (Phenomenex, Torrance, Calif.). Protein was eluted byapplying a linear gradient from 0-80% acetonitrile in 0.1% TFA. Duringthis elution, both a major and minor protein peak was detected by UVabsorbance at 210 nm and 277 run (FIG. 1 Panel (A)). The two separatefractions were lyophilized to dryness and resuspended in 50 μL ofdenaturation buffer (8M Urea, 0.4 M ammonium bicarbonate, pH 7.5). Bothprotein fractions were digested with modified trypsin for approximately18 hours at 37° C. Trypsin was added at a 1:40 ratio (w/w) relative toeach sulfatase fraction. Prior to proteolysis, cysteines were firstsubjected to reductive carboxymethylation by the addition of 5 mMdithiothreitol for 1 hour at 50° C., followed by the addition of 20 mMiodoacetic acid for 30 minutes (room temperature). The alkylationreaction was quenched by the addition of 50 μL denaturation buffer. Theresulting peptides were resolved by RP-HPLC on a 250 mm×2 mm C4 columnusing a linear gradient of 2-80% acetonitrile in 0.1% trifluoraceticacid carried out over a 120 minute timecourse. Select peptidescorresponding to chromatography peaks 2, 3, 4, 5, and 8 (FIG. 1 Panel(B)) were sequenced using an on-line Model 120phenylthiohydantoin-derivative analyzer (Biopolymers Laboratory,Massachusetts Institute of Technology).Molecular cloning of theflavobacterial 2-O sulfatase—The 2-O sulfatasewas cloned from a λZAP II flavobacterial genomic library constructed andscreened essentially as described for the Δ 4,5 glycuronidase (Myette,J. R., Shriver, Z., Kiziltepe, T., McLean, M. W., Venkataraman, G., andSasisekharan, R. (2002) Biochemistry 41(23), 7424-7434). A 600 base pairDNA plaque hybridization probe was generated by PCR using degenerateprimers 5′ ATHGAYATHATHCCNACNATH 3′ (forward, SEQ ID NO: 8) and 5′DATNGTYTCATTNCCRTGYTG 3′ (reverse, SEQ ID NO: 9). PCR was carried outfor 35 cycles using a 52° C. annealing temperature and 2 minuteextensions at 72° C. The specificity of this probe was established byDNA sequence analysis, which indicated a direct correspondence of itstranslated sequence to peak 1 tryptic peptides. Based on thisinformation, the non-degenerate primers 5′ CATACACGTATGGGCGATTAT 3′(forward, SEQ ID NO: 10) and 5′ GATGTGGGGATGATGTCGAT 3′ (reverse, SEQ IDNO: 11) were subsequently used in place of the original degenerateprimers. PCR amplified DNA probe was gel purified and subsequently ³²Pradiolabeled using the Prime-it II random priming kit (Stratagene).Plaques were lifted on to nylon membranes (Nytran Supercharge,Schleicher and Schuell, Keene, N.H.) and DNA was crosslinked to eachfilter by UV-irradiation. Plaque hybridizations were completed overnightat 42° C. according to standard methods and solutions (Current Protocolsin Molecular Biology (1987) (Ausubel, F. M., Brent, R., Kingston, R. E.,Moore, D. D., Seidman, J. G., Smith, J. A., and Struhl, K., Ed.) 1-3vols., John Wiley and Sons, New York). Positive clones were visualizedby phosphor imaging (Molecular Dynamics, Piscataway, N.J.) and/or ³²Pautoradiography. Clones were further purified by secondary and tertiaryscreens and the recombinant phage was excised as a double-strandedphagemid (pBluescript) as described by the manufacturer (Stratagene).Recombinants were confirmed by DNA sequencing using both T7 and T3primers. Insert size was determined by restriction mapping ofpBluescript inserts using Not 1, Xba 1, and Xho 1.

The full-length sulfatase gene (phagemid clone S4A) was subcloned intothe T7-based expression plasmid pET28a in three steps. In the first PCRstep, Nde 1 and Xho I restriction sites were introduced at the 5′ and 3′termini of the 2-O sulfatase coding sequence by using primers 5′TGTTCTAGACATATGAAGATGTACAAATCGAAAGG 3′ (SEQ ID NO: 12) and 5′GTCTCGAGGAT CCTTATTTTTTTAATGCATAAAACGAATCC 3′ (SEQ ID NO: 13),respectively. At the same time, the Nde 1 restriction site alreadypresent within the sulfatase gene starting at position 1049 (FIG. 2) wasabolished by silent mutagenesis (CATATG→CATCTG) using the mutagenicprimers 5′ GATATTATCCCCACCATCTGTGGCTTTGCCGGAA 3′ (SEQ ID NO: 14) and 5′TTCCGGCAAAGCCACAGA TGGTGGGGATAATATC 3′ (SEQ ID NO: 15), with the A to Ctransversion noted in bold. In the second step, the final PCR productwas gel purified and ligated into the TOPO/TA PCR cloning vector pCR 2.1(Invitrogen) following the addition of 3′ dA overhangs with 0.5 units ofTaq polymerase and 300 μM dATP (10 minutes, 72° C.). Ligated DNA wastransformed into One-shot TOP10 chemically competent cells. Positiveclones were identified by blue/white colony selection and confirmed byPCR colony screening. In the third step, the 1.5 kb sulfatase gene wasexcised from pCR 2.1 TOPO and pasted into pET28a (Novagen, Madison,Wis.) as an Nde 1-Xho 1 cassette. Final expression clones were confirmedby plasmid DNA sequencing.

A 2-O sulfatase amino terminal truncation lacking the first 24 aminoacids (2-O ΔN¹⁻²⁴) was PCR cloned as above except the forward primer 5′TCTAGACATATGCAAACCTCAAAA GTAGCAGCT 3′ (SEQ ID NO: 16) was used in placeof original outside 5′ primer listed. In this DNA construct, the 2-Osulfatase-specific sequence begins with Q25 (FIG. 2) and readsMQTSKVAASRPN (SEQ ID NO: 17).

Recombinant Expression and protein purification of a 6× histidine-tagged2-O sulfatase (and 2-O ΔN¹⁻²⁴)—Both the full-length enzyme and thetruncated enzyme (2-O ΔN¹⁻²⁴) were recombinantly expressed in the E.coli strain BL21 (DE3) (Novagen) initially as NH₂-terminal 6× histidinefusion proteins to facilitate purification. The protocol for theirexpression and subsequent one-step purification by nickel chelationchromatography was as previously described for the Δ 4,5 glycuronidase(Myette, J. R., Shriver, Z., Kiziltepe, T., McLean, M. W., Venkataraman,G., and Sasisekharan, R. (2002) Biochemistry 41(23), 7424-7434). Greaterthan 90% of the enzyme was eluted from a 5 ml column in a single 12.5 mlfraction following the addition of high imidazole elution buffer (50 mMTris-HCL, pH 7.9, 0.5 M NaCl, and 250 mM imidazole). The enzyme wasimmediately diluted with 2 volumes of cold enzyme dilution buffer (50 mMTris, pH 7.5, 100 mM NaCl). Cleavage of the 6X histidine tag by thrombinwas achieved by the step-wise addition of 10 units of biotinylatedthrombin (total 50 units) to 30 mL of diluted enzyme over the course ofseveral hours while gently mixing by inversion at 4° C. Substantialprecipitation of the sulfatase routinely occurred during the cleavagereaction. Thrombin was recovered by the addition of streptavidin agaroseusing the thrombin cleavage capture kit (Novagen). Capture was carriedout a 4° C. for 2 hours with gentle mixing. Bound thrombin was collectedby centrifugation for 5 minutes at 500×g. Supernatant containing soluble2-O sulfatase was then dialyzed at 4° C. against 12 liters of enzymedilution buffer using 20.4 mm diameter Spectra/Por dialysis tubing(Spectrum Laboratories, Rancho Dominguez, Calif.) with a 10,000 MWCO.Following dialysis, the purified sulfatase was concentrated using aCentriplus YM10 ultrafiltration device (Millipore, Watertown, Mass.).The enzyme was stable for at least two weeks at 4° C. Long-term storagewas carried out at −85° C. in the presence of 10% glycerol without anysubsequent loss of activity due to freezing and thawing.

Protein concentrations were determined by the Bio-Rad protein assay andconfirmed by UV spectroscopy using a theoretical molar extinctioncoefficient (ε280) of 77,380 M⁻¹ for 2-O ΔN¹⁻²⁴ with the histidine tagremoved. Protein purity was assessed by silver-staining ofSDS-polyacrylamide gels.

Computational methods—Sulfatase multiple sequence alignments were madefrom select BLASTP database sequences (with scores exceeding 100 bitsand less than 6% gaps) using the CLUSTALW program (version 1.81) presetto an open gap penalty of 10.0, a gap extension penalty of 0.20, andboth hydrophilic and residue-specific gap penalties turned on. Signalsequence predictions were made by SignalP V1.1 using the von Heijnecomputational method (Nielsen, H., Engelbrecht, J., Brunak, S., and vonHeijne, G. (1997) Protein Eng 10(1), 1-6).Molecular mass determinations by MALDI-MS—The molecular weight of the2-O sulfatase NH₂ truncated enzyme (2-O ΔN¹⁻²⁴) was determined bymatrix-assisted laser desorption ionization mass spectrometry (MALDI-MS)essentially as described (Rhomberg, A. J., Ernst, S., Sasisekharan, R.,and Biemann, K. (1998) Proc Natl Acad Sci USA 95(8), 4176-81). TheNH₂-terminal histidine tag of the recombinant protein was cleaved bythrombin prior to mass analysis. 1 μL of a 2-O sulfatase solution(diluted in water to 0.5 mg/mL) was added to 1 μl of a saturatedsinapinic acid matrix solution previously deposited onto the plate. Theobserved mass of the recombinant enzyme was corrected according to anexternal calibration using mass standards.2-O sulfatase assay and determination of biochemical reactionconditions—2-O sulfatase activity was measured using the unsaturatedheparin trisulfated disaccharide ΔU_(2S)H_(NS,6S) or the disulfateddisaccharide ΔU_(2S)H_(NS) as well as the disulfated disaccharideΔUH_(NS,6S) lacking a sulfate at the 2-OH position. Standard reactionsincluded 50 mM imidazole, pH 6.5, 50 mM NaCl, 500 μM disaccharide, and25 nM of enzyme (2-O ΔN¹⁻²⁴) in a 20 μL reaction volume. The reactionwas carried out for 30 seconds at 30° C. Prior to its addition, theenzyme was serially diluted to 250 nM in ice cold 1× imidazole buffer.The assay was initiated by the addition of 2 μL of this 10× enzyme stockto 18 μL of reaction mixture. The enzyme was inactivated by heating at95° C. 11 for five minutes in pre-heated 0.5 mL eppendorf tubes.Desulfation at the 2-OH position of the disaccharide was measured bycapillary electrophoresis. Resolution of substrate and product wereachieved under standard conditions described for HSGAG compositionalanalyses (Rhomberg, A. J., Ernst, S., Sasisekharan, R., and Biemann, K.(1998) Proc Natl Acad Sci USA 95(8), 4176-81). Activity was generallymeasured as moles of desulfated product formed and was calculated fromthe measured area of the product peak based on molar conversion factorsempirically determined from standard curves. For the detection of mono-and di-sulfated disaccharide products, total electrophoresis time was 20minutes. Each unsaturated disaccharide peak was detected by UVabsorption at 232 nm.

For pilot experiments measuring the relative effect of ionic strength on2-O sulfatase activity, the NaCl concentration was varied from 0.05 to 1M in 50 mM MES buffer (pH 6.5) that included 500 μM of the disulfateddisaccharide ΔU_(2S)H_(NS,6S) and 50 nM enzyme. The effect of pH onsulfatase activity was assessed as a function of catalytic efficiency bymeasuring kinetic parameters in the following two overlapping pH buffersystems ranging from 5.0 to 8.0: 50 mM MES at pH 5.0, 5.5, 6.5, and 7.0;50 mM MOPS at pH 6.5, 7.0, 7.5 and 8.0. Assays included 25 nM enzyme, 50mM NaCl and varying concentrations of the disulfated disaccharidesubstrate ΔU_(2S)H_(NS). K_(m) and k_(cat) values were extrapolated fromV_(o) vs. [S] curves fit to the Michaelis-Menten equation by anon-linear least squares regression and the relative k_(cat)/K_(m)ratios plotted as a function of buffer pH. Based on this profile,relative enzyme activity was also measured in four different buffers(MES, imidazole, ADA, and sodium phosphate) each present as a 50 mMconcentration at pH 6.5. Relative activities were measured at a singlesaturating substrate concentration (4 mM) using ΔU_(2S)H_(NS).

Tandem use of 2-O sulfatase and Δ 4,5 glycuronidase in HSGAGcompositional analyses—200 μg of heparin was first digested with allthree heparinases in an overnight digestion in glycuronidase reactionbuffer which included 50 mM PIPES, pH 6.5, 50 mM NaCl and a 100 μLreaction volume. The heparinase digestion mix was split into 4×20 μLreactions which were individually treated as follows: Tube 1, noaddition (heparinase only control); Tube 2, 5 μg of Δ 4,5 glycuronidase,30° C. 1 hour; Tube 3, 5 μg 2-O sulfatase (2-O ΔN¹⁻²⁴) 37° C., 1 hour;Tube 4, 2-O sulfatase and Δ 4,5 glycuronidase added simultaneously, 30°C., 1 hour. Δ 4,5 glycuronidase activity was ascertained by adisappearance of unsaturated disaccharide peaks due to the loss of UVabsorption at 232 nm.

The substrate-product relationship between the two enzymes was examinedby directly measuring Δ 4,5 glycuronidase activity either before orfollowing the addition of recombinant 2-O sulfatase. Reactions werecarried out at 30° C. and included 50 mM MES, pH 6.5, 100 mM NaCl, and 2mM ΔU_(2S)H_(NS) in a 100 μL reaction volume. In these experiments, 250nM Δ 4,5 glycuronidase and 25 nM 2-O ΔN¹⁻²⁴ were sequentially added asfollows: Δ 4,5 alone, Δ 4,5 followed by 2-O sulfatase, or 2-O sulfatasefollowed by Δ 4,5. In each case, the first enzyme was added to thereaction in a 2 minute preincubation step. Δ 4,5 glycuronidase activitywas measured immediately following the addition of the second enzyme bydetermining the rate of substrate disappearance as monitored by the lossof UV absorption at 232 nm (Myette, J. R., Shriver, Z., Kiziltepe, T.,McLean, M. W., Venkataraman, G., and Sasisekharan, R. (2002)Biochemistry 41(23), 7424-7434). Δ 4,5 activity for the corresponding2-O desulfated disaccharide ΔUH_(NS) was also measured under identicalconditions.

Homology modeling of 2-O sulfatase—The crystal structure of humanarylsulfatase A, human arylsulfatase B, and the P. aeruginosaarylsulfatase (von Bulow, R., Schmidt, B., Dierks, T., von Figura, K.,and Uson, I. (2001) J Mol Biol 305(2), 269-77) were used to obtain astructural model for the 2-O sulfatase enzyme. A multiple sequencealignment was performed using CLUSTALW algorithm (Higgins, D. G.,Thompson, J. D., and Gibson, T. J. (1996) Methods Enzymol 266, 383-402)on the 2-O sulfatase and the sulfatase sequences whose crystalstructures have been solved (human arylsulfatase A, B and P. aeruginosaarylsulfatase) (FIGS. 9 and 16). Based on this multiple sequencealignment, three model structures of 2-O sulfatase were obtainedcorresponding to its alignment with the other three sulfatases. Themodels were constructed using the Homology module of Insight IImolecular simulations package (Accelrys, San Diego, Calif.). The sidechain of the critical Cys 82 which is shown to undergo posttranslationalmodification in the active enzyme was replaced by the geminal diol[C_(β)(OH)₂]. The potentials for the model structures were assignedusing the AMBER force field (Homans, S. W. (1990) Biochemistry 29(39),9110-8). The deletions in the modeled structure were closed using 200steps of steepest descent minimization without including charges bykeeping most of the structure rigid and allowing the regions close tothe deletion move freely. The final refined structure was subjected to400 steps of steepest descent minimization without including charges and400 steps of conjugate gradient minimization including charges.Molecular docking of disaccharide substrates into the active site of themodeled 2-O sulfatase—Heparin derived disaccharides with a ΔU at thenon-reducing end were modeled as follows. The coordinates of thetrisulfated ΔU containing disaccharide (ΔU_(2S)H_(NS,6S)) were obtainedfrom the co-crystal structure of a heparinase derived hexasaccharidewith fibroblast growth factor 2 (PDB id: 1BFC). This trisulfateddisaccharide structure was used as a reference to generate thestructural models for other disaccharides including ΔU_(2S)H_(NS),ΔU_(2S)H_(NAc) and ΔU_(2S)H_(NAc,6S). The coordinates of trisulfateddisaccharides (I_(2S)H_(NS,6S)) containing iduronic acids in the ¹C₄ and²S₀ conformations were also obtained from 1BFC (PDB id: 1BFC). Similarlychondroitin sulfate derived disaccharides ΔU_(2S)Gal_(NAc,4S) andΔU_(2S)Gal_(NAc,6S) were modeled using a reference structure of achondroitin-4 sulfate disaccharide ΔUGal_(NAc,4S) whose coordinates wereobtained from its co-crystal structure with the chondroitinase B enzyme(PDB id: 1DBO). The potentials for these disaccharides were assignedusing the AMBER force field modified to include carbohydrates (Homans,S. W. (1990) Biochemistry 29(39), 9110-8) with sulfate and sulfamategroups (Huige, C. J. M., Altona, C. (1995) J. Comput. Chem. 16, 56-79).

The orientation of the cleavable sulfate group relative to Oγ1 of thegeminal diol in the active site of human arylsulfatase A and thebacterial arylsulfatase was identical as observed in their respectivecrystal structures. This orientation was such that one of the faces ofthe tetrahedral formed by the 3 oxygen atoms of SO₃ ⁻ was orientedtowards Oγ1 facilitating the nucleophilic attack of the sulfur atom andthe transfer of the SO₃ ⁻ group to Oγ1 (Waldow, A., Schmidt, B., Dierks,T., von Bulow, R., and von Figura, K. (I 999) J Biol Chem 274(18),12284-8). This highly specific orientation of the sulfate group helpedin positioning the disaccharide substrates relative to the active siteof the 2-O sulfatase. After fixing the orientation of the 2-O sulfategroup, the glycosidic torsion angles and exocyclic torsion angles wereadjusted manually to remove unfavorable steric contacts with the aminoacids in the active site. The enzyme substrate complexes were minimizedusing 200 steps of steepest descent followed by 400 steps ofNewton-Raphson minimization including charges. Most of the enzyme waskept rigid and only the loop regions constituting the active site wereallowed to move freely. To model the disaccharide structure, a forcingconstant of 7000 kcal/mole was applied to the ring torsion angles duringthe energy minimization calculations while simultaneously fixing thering conformation of the individual monosaccharide units. The manualpositioning of the substrates was done using the Viewer module, buildingof the disaccharide structures from the reference structures was, doneusing the Builder module and the energy minimization was done using theDiscover module of Insight II.

Heparin compositional analyses by capillary electrophoresis andMALDI-MS—Approximately 10 μg of the AT-10 oligosaccharide were incubatedwith 100 picomoles of 2-O ΔN¹⁻²⁴ in a 40 μL reaction volume at 30° C. 15μL aliquots were removed at 4 hours and 17 hours and heat inactivated at95° C. The oligosaccharide reaction products (along with 15 μL of aminus sulfatase control) were subjected to an exhaustive heparinase Iand III digestion prior to CE-based compositional analysis. Desulfationof the decasaccharide was assayed in parallel by MALDI-MS usingestablished methods (Rhomberg, A. J., Ernst, S., Sasisekharan, R., andBiemann, K. (1998) Proc Natl Acad Sci USA 95(8), 4176-81.).Substrate specificity and kinetics experiments using differentdisaccharide substrates—For substrate specificity experiments, thefollowing heparin disaccharide substrates were used: ΔU_(2S)H_(NAc),ΔU_(2S)H_(NAc,6S), ΔU_(2S)H_(NS), and ΔU_(2S)H_(NS,6S). In addition, thechondroitin disaccharides ΔU_(2S)Gal_(NAc,4S) and ΔU_(2S)Gal_(NAc,6S)were also studied. Disaccharide concentrations for each respectivesubstrate were varied from 0.1 mM to 4 mM. Initial rates (V_(o)) wereextrapolated from linear activities representing <20% substrate turnoverand fit to pseudo first-order kinetics. Standard reactions included 50mM imidazole, pH 6.5, 50 mM NaCl, 500 μM disaccharide, and 25 nM ofenzyme (2-O ΔN¹⁻²⁴) in a 20 μL reaction volume. The reaction was carriedout for 30 seconds at 30° C. Prior to its addition, the enzyme wasserially diluted to 250 nM in ice cold 1× imidazole buffer. The assaywas initiated by the addition of 2μL of this 10× enzyme stock to 18 μLof reaction mixture. Sulfatase activity was inactivated for five minutesat 95° C. in pre-heated 0.5 mL eppendorf tubes. Desulfation at the 2-OHposition of the disaccharide was measured by capillary electrophoresis.Resolution of substrate and product were achieved under standardconditions described for HSGAG compositional analyses (Venkataraman, G.,Shriver, Z., Raman, R., and Sasisekharan, R. (1999) Science 286(5439),537-42). Activity was measured as moles of desulfated product formed andwas calculated from the measured area of the product peak based on molarconversion factors empirically determined from standard curves. For thedetection of mono- and di-sulfated disaccharide products, totalelectrophoresis time was 25 minutes. Each unsaturated disaccharide peakwas detected by UV absorption at 232 nm. All the substrate saturationkinetics were measured under Michaelis-Menten conditions.2-O sulfatase active site labeling and peptide mapping—Approximately 500μg of 6× histidine-tagged 2-O ΔN¹⁻²⁴ (wild-type enzyme and C82Asite-directed mutant) were lyophilized by Speed-Vac centrifugation andvigorously resuspended in 90 μL denaturation buffer containing 6Mguandinium hydrochloride, 0.1 M Tris-HCL, pH 7.5. Active site aldehydeswere fluorescently labeled by adding 25 μL of Texas Red hydrazine madeup as a 10 mM stock in dimethyl formamide (DMF). Labeling was carriedout for three hours at room temperature with gentle mixing on a rotatingplatform. The hydrazone linkage was stabilized by the addition of 10 μLof a fresh 5M sodium cyanoborohydride stock made up in 1N NaOH.Reduction was carried out for 1 hour at room temperature. Unreactedfluorophore was removed by repeated acetone precipitation (added 5:1v:v). Acetone was prechilled at −20° C. Samples were chilled at −85° C.for 20 minutes prior to spinning in a microfuge for 10 minutes, maximumspeed, at 4° C. Pellets were briefly dried by Speed-Vac centrifugation.

The labeled sulfatase (and unlabeled control) were proteolyzed withsequence grade-modified trypsin for 20 hours at 37° C. in digestionbuffer that contained 0.1 M Tris-HCL, pH 8.5, 1 mM EDTA, 1 mM DTT and10% acetonitrile (v/v) in a 30 μL reaction volume. Trypsin was firstreconstituted as a 2.5 mg/mL stock in 1% acetic acid and added at a 1:5ratio (w/w) relative to the target protein. Following trypsin digestion,peptide cysteines were reduced by the addition of 50 mM DTT (50° C.under argon, 1 hour). Reduced cysteines were subsequently alkylated for30 minutes at 37° C. (in the dark) by the addition of 150 mMiodoacetamide, added from a 2M stock made up in 0.1M Tris-HCL, pH 8.5.This reduction-alkylation cycle was repeated one more time.

Molecular masses of select peptides were determined by MALDI-MS asdescribed (Myette, J. R., Shriver, Z., Liu, J., Venkataraman, G.,Rosenberg, R., and Sasisekharan, R. (2002) Biochem Biophys Res Commun290(4), 1206-13) using 1 μL of α-cyano-4-hydroxycinnamic acid (CHCA) in50% acetonitrile, 0.3% TFA as a matrix.

Site-directed mutagenesis of the C82A active site mutan—Thesite-directed mutant C82A was cloned by recombinant PCR using outsideprimers 5′ TCT AGA CAT ATG CAA ACC TCA AAA GTA GCA GCT 3′ (forward, SEQID NO: 18) and (5′ GT CTC GAG GAT CCT TAT TTT TTT AAT GCA TAA AAC GAATCC 3′ (reverse, SEQ ID NO: 19) in addition to the following mutagenicprimer pair: 5′ CCAG CCG CTC GCT ACA CCT TCA CG 3′ (forward, SEQ ID NO:20) and 5′CG TGA AGG TGT AGC GAG CGG CTG G 3′ (reverse, SEQ ID NO: 21).The engineered codon change for each DNA strand is underlined.Subcloning into pET28a, recombinant expression in the E. coli strainBL21 (DE3), and subsequent purification by nickel chelationchromatography using the N-terminal 6× histidine purification tag are asdescribed above for 2-O ΔN¹⁻²⁴.Circular dichroism—Recombinantly expressed 2-O sulfatase and theinactive C82A mutant were concentrated and buffer-exchanged into 50 mMsodium phosphate, pH 7.0, using a Centricon 10 ultrafiltration device(Millipore). CD spectra were collected on an Aviv 62DSspectropolarimeter equipped with a thermostatic temperature control andinterfaced to an IBM microcomputer. Measurements were performed in aquartz cell with a 1 mm path length. Spectra were recorded at 25° C. inan average of 5 scans between 205 and 270 nm with a 1.0 nm bandwidth anda scan rate of 12 nm/min. CD band intensities are expressed as molarellipticities, θM, in degrees·cm²-dmol⁻¹.ResultsMolecular cloning and recombinant expression of the F. heparinum 2-Osulfatase—As a first step towards the cloning the 2-O sulfatase gene, wepurified the enzyme directly from the native bacterium followed by apartial determination of its amino acid sequence. After a five-stepchromatographic fractionation of flavobacterial lysates, we achieved agreater than 3000-fold purification of sulfatase activity. Furtherfractionation of this activity by reverse phase HPLC chromatographyyielded two separate polypeptides (FIG. 1, Panel (A)). Both proteinswere subjected to a limit trypsin digestion and the resultant peptideslikewise purified by reverse phase HPLC (FIG. 1, Panel (B)). From selectpeak 1 peptide sequences, degenerate primers were synthesized. Weinitially screened primer pairs corresponding exclusively to peak 1protein sequence (Table 1), given the fact that this sulfatase fractionrepresented the major protein species present in the final purificationstep. PCR amplification of genomic DNA using degenerate primerscorresponding to peptide peaks 3 and 5 yielded a discrete 600 bp DNAproduct. Sequence analysis of this amplified DNA indicated a translatedamino acid sequence to which three of the isolated peak I peptidesmapped. We used this DNA, therefore, as a hybridization probe to screena λZAP flavobacterial genomic library and isolate a full-length clone.Several positive clones were isolated; most of them contained an averageinsert size between 4-5 kb. One genomic clone of approximately 7 kb(clone S4A) was subjected to direct DNA sequencing. This clone containedat least one open reading frame (ORF) in particular that encodes aputative protein of 468 amino acids in length (464 amino acids fromfirst methionine) and whose primary sequence includes all of thesulfatase peptides for which we had obtained sequence information (FIG.2). Based on its amino acid composition, the encoded protein is quitebasic (theoretical pI of 8.75), with 67 basic side chains comprising 14approximately 14% on a molar basis. The putative sulfatase alsopossesses 8 cysteines in addition to 46 aromatic amino acids.

TABLE 1 2-O-sulfatase peptides and corresponding degenerate primers PeakNo. Peptide Sequence Degenerate Primers 2 YIVYDKGEIR5′ TAYATHGTNTAYGAYAARGG 3′ (SEQ ID NO:22) (SEQ ID NO:27)5′ NCCYTTRTCRTANACDATRTA 3′ (SEQ ID NO:28) 3 TYPSVGWNESQWR5′ CARCAYGGNTTYGARACNAT 3′ (SEQ. ID. NO:23) (SEQ ID NO:29)5′ DATNGTYTGATTNCCRTGYTG 3′ (SEQ ID NO:30) 4KMPHETGFTGNTPEKDGQWPDSVLMMGK 5′ TAYATHGTNTAYGAYAARGG 3′ (SEQ ID NO:24)(SEQ ID NO:31) 5′ NCCYTTRTANACDATRTA 3′ (SEQ ID NO:32) 5VAQHGFETIENTGMGDYTDAVTPSQCANFNK 5′ ATHGAYATHATHCCNACNAT 3′ (SEQ IDNO:25) (SEQ ID NO:33) 5′ DATNGTNGGDATDATRTCDAT 3′ (SEQ ID NO:34) 8TDDQLVCNGIDIIPTICGFAGIAK 5′ GAYATHATHCCNACNATHTGYTT 3′ (SEQ ID NO:26)(SEQ ID NO:35) 5′ AARCADATNGTNGGDATDATRTC 3′ (SEQ ID NO:36) SelectRP-HPLC purified tryptic peptides (see also FIG. 1, Panel (B)) weresubjected to amino acid sequencing. Also shown are the correspondingdegenerate primers.

Upon a closer examination of its primary sequence, we also identified aconserved sulfatase domain. This signature domain included the consensussequence C/SXPXRXXXXS/TG (SEQ ID NO: 6) presumably comprising (at leastin part) the sulfatase active site and possessing the cysteine (denotedin bold) that is most likely modified as a formylglycine in vivo. Theputative 2-O sulfatase that we cloned from F. heparinum exhibitssubstantial homology to many members of a highly conserved sulfatasefamily (FIG. 3) (Bond, C. S., Clements, P. R., Ashby, S. J., Collyer, C.A., Harrop, S. J., Hopwood, J. J., and Guss, J. M. (1997) Structure5(2), 277-89, Parenti, G., Meroni, G., and Ballabio, A. (1997) Curr OpinGenet Dev 7(3),386-91). A structurally-oriented description of thishomology and its correlation to enzyme function is found below.

From this sequence information, we were confident that we had indeedcloned a sulfatase from the flavobacterial genome. To ultimatelyestablish its functionality, we next set out to recombinantly expressthis protein in E. coli. The full-length gene (beginning at the firstmethionine noted in FIG. 2) was subdloned into the T7-based expressionvector, pET28a for expression as an NH₂-terminal 6× histidine-taggedprotein to facilitate purification. Induction with IPTG led to a limitedsoluble expression of a polypeptide whose apparent molecular weightroughly corresponded to the theoretical mass of the fusion protein(approximately 54 kDa). Using Ni⁺² chelation chromatography, we wereable to partially purify this polypeptide from the bacterial lysate andunequivocally measure 2-O specific sulfatase activity using thetrisulfated, unsaturated heparin disaccharide ΔU_(2S)H_(NS,6S) as asubstrate.

We identified a putative signal sequence for the flavobacterial 2-Osulfatase comprised of the first 24 amino acids (see FIG. 2). Byengineering a 2-O sulfatase N-terminal truncation lacking this sequence(herein referred to as 2-O ΔN¹⁻²⁴), we achieved high expression levelsof soluble, highly active enzyme. Protein yields exceeding 100 mg ofrelatively pure sulfatase per liter of induced bacterial cultures wereroutinely achieved using a single chromatographic step (FIG. 4). Thespecific activity of the recombinant sulfatase was considerably enhancedfollowing the removal of the N-terminal 6×-histidine tag by thrombincleavage. Removal of this purification tag resulted in a greater than10-fold purification of sulfatase activity relative to the crudebacterial lysate (Table 2). For this reason, we used the cleaved proteinin all subsequent experiments. The molecular weight of thisrecombinantly expressed sulfatase as determined by MALDI-MS is 50,120.8Daltons. This empirical value closely agrees with its theoretical massof 49,796 Daltons that is based entirely on its amino acid composition.

TABLE 2 Purification of recombinant 2-O-sulfatase Specific ActivityTotal Protein (nanomoles of Fold- Fraction (mg) DiS/min/μg protein)purification lysate 322 4.14 — Ni⁺² (with His Tag) 122 7.43 1.8 His Tagremoval  15* 42.3 10.2 200 ng of total protein from each purificationstep was assayed for 2-O-sulfatase activity as described in Materialsand Methods using the unsaturated heparin disaccharide (DiS)U_(2S)H_(NS) as a substrate. Fold purification is relative to crudebacterial lysate. *Soluble enzyme remaining after substantial loss dueto protein precipitation.

To establish the recombinant enzyme's exclusivity for the uronic acid2-O sulfate, we initially compared two related unsaturated heparindisachharides: ΔU_(2S)H_(NS,6S) versus ΔUH_(NS,6S). The recombinantsulfatase only hydrolyzed a single sulfate, namely, the one found at the2-OH position (FIG. 5).

Biochemical conditions for optimal in vitro activit—Having successfullyachieved the recombinant expression and purification of theflavobacterial sulfatase as a soluble enzyme as well as demonstration ofits unequivocal specificity for the uronic acid 2-O sulfate, we next setout to define the reaction conditions required for optimal enzymeactivity in vitro. These parameters included pH, temperature, ionicstrength, and possible divalent metal ion dependency. In brief, theenzyme exhibited a pH activity range between 6.0 and 7.0, with optimumactivity occurring at pH 6.5 (FIG. 6, Panel (A)). The enzyme wasessentially inactive at the outlying pH values of 5.0 and 8.0. In termsof different buffer systems (all at pH 6.5), an imidazole-based bufferdemonstrated the highest relative activity as compared with bufferscontaining 50 mM MES, ADA, or phosphate. As expected, phosphate bufferwas clearly inhibitory (FIG. 6, Panel (A) inset).

We also examined 2-O sulfatase activity relative to ionic composition.The recombinant enzyme was optimally active at approximately 50 mM NaCl.Activity was sharply inhibited by [NaCl] exceeding 100 mM, with 50%inhibition occurring at less than 250 mM NaCl (FIG. 6, Panel (B)).Maximal enzyme activity was largely unaffected by the addition of EDTAup to a 1 mM concentration. Addition of exogenous CaCl₂, MgCl₂, or MnCl₂(up to 10 mM) also had no substantive effect, indicating that theseparticular divalent metal ions are not required. A preincubation of theenzyme with 5 mM EDTA did result in an approximately 10% inhibition ofactivity using the trisulfated disaccharide as a substrate.

37° C. was the default temperature at which all of the preliminarybiochemical experiments were conducted. We measured both relative enzymeactivity and stability as a function of varying reaction temperature(FIG. 6, Paenl (C)). The 2-O sulfatase was active over a fairly broadtemperature range (25° C. to 37° C.), with optimal activity occurring at30° C. Enzyme activity was compromised at 42° C. Enzyme stability atthis temperature was likewise affected as assessed in pre-incubationexperiments conducted at varying temperatures (30° C.→42° C.) prior tomeasuring 2-O sulfatase activity at 30° C.

The substrate-product relationship between the 2-O sulfatase and Δ 4,5glycuronidase—As we have already noted, the flavobacterial Δ 4,5glycuronidase is unable to hydrolyze unsaturated saccharides possessinga uronic acid 2-O sulfate at the non-reducing end (Myette, J. R.,Shriver, Z., Kiziltepe, T., McLean, M. W., Venkataraman, G., andSasisekharan, R. (2002) Biochemistry 41(23), 7424-7434). We hypothesizedthat, an obligatory substrate-product relationship between the 2-Osulfatase and the Δ 4,5 glycuronidase may exist. We examined a possiblekinetic relationship between these two enzymes by looking at theirsequential action (FIG. 7). In this experiment, Δ 4,5 glycuronidaseactivity was measured directly either during or following the additionof the recombinant 2-O sulfatase using the disaccharide substrateΔU_(2S)H_(NS). When this disaccharide was incubated with the Δ 4,5enzyme alone, it was completely refractory to glycuronidase-mediatedhydrolysis as measured by a loss of absorbance at 232 nm. A 2 minutepreincubation of the substrate with the 2-O sulfatase, however resultedin robust linear glycuronidase activity. This rate was comparable to therate of hydrolysis measured for the control substrate ΔUH_(NS) using theΔ 4,5 enzyme alone. In the reciprocal experiment (i.e., whereby the 2-Osulfatase was added second), we observed an initial lag in Δ 4,5activity. This lag was followed by a linear Δ 4,5 activity, albeit at aslower rate than in the case where the 2-O sulfatase was added first.The observed delay in activity was presumably due to the prerequisite2-O desulfation of the substrate which must occur prior to being actedon by the glycuronidase. This experiment clearly demonstrates at least afunctional linkage between these two HSGAG degrading enzymes.

With the results just described, we considered the parallel use of thesetwo enzymes (along with the heparinases) as complementary tools forHSGAG compositional analyses. The utility of this combinatorial approachis shown in FIG. 8. 200 μg of heparin were first subjected to anexhaustive heparinase treatment. Subsequent treatment of the cleavageproducts with the Δ 4,5 glycuronidase resulted in the disappearance ofselect saccharide peaks, namely those that did not possess a 2-Osulfated uronic acid at the non-reducing end (FIG. 8, Panel (B)).Conversely, subsequent treatment of the heparinase-derived saccharideswith the 2-O sulfatase results in both the disappearance of 2-O sulfateddisaccharides as well as a concomitant appearance of their desulfatedproducts (FIG. 8, Panel (C)). When both the Δ 4,5 glycuronidase and the2-O sulfatase were added simultaneously to the heparinase cleavageproducts, essentially all of the saccharides were hydrolyzed by the A4,5 glycuronidase as evident by a lack of any UV absorbableelectrophoresis products (FIG. 8 Panel (D).

Structure-based homology modeling of the 2-O sulfatase active site—Thecrystal structures of three sulfatases have been solved. Thesesulfatases are human arylsulfatase A (Lukatela, G., Krauss, N., Theis,K., Selmer, T., Gieselmann, V., von Figura, K., and Saenger, W. (1998)Biochemistry 37(11), 3654-64, von Bulow, R., Schmidt, B., Dierks, T.,von Figura, K., and Uson, I. (2001) J Mol Biol 305(2), 269-77),arylsulfatase B (N-acetylgalactosamine-4-sulfatase) (Bond, C. S.,Clements, P. R., Ashby, S. J., Collyer, C. A., Harrop, S. J., Hopwood,J. J., and Guss, J. M. (1997) Structure 5(2), 277-89), and a bacterialarylsulfatase from Pseudomonas aeruginosa (Boltes, I., Czapinska, H.,Kahnert, A., von Bulow, R., Dierks, T., Schmidt, B., von Figura, K.,Kertesz, M. A., and Uson, I. (2001) Structure (Camb) 9(6), 483-91). Incomparing their structures, we observed a structural homology betweeneach of them, especially as it pertained to a conservation of criticalactive site residues and their spatial arrangement. By extension, mostof these amino acids were likewise conserved in the flavobacterial 2-Osulfatase as evident by a direct alignment of their primary sequences(FIGS. 9 and 16). We used this close structural relationship toconstruct three homology-based models for the flavobacterial 2-Osulfatase, each one based on one of the three crystal structuresexamined. We ultimately chose as our representative 2-O sulfatasestructure the homology model constructed using theN-acetylgalactosamine-4-sulfatase (arylsulfatase B) (FIG. 10). Thisdecision was largely based on it also being a GAG desulfating enzyme. Inthis model, we replaced cysteine 82 with a formylglycine (FGly 82). Wechose to represent FGly 82 in the hydrated state as a geminal diol[-C_(β)(OH)₂], consistent with the proposed resting state (beforecatalysis) of the enzyme (Lukatela, G., Krauss, N., Theis, K., Selmer,T., Gieselmann, V., von Figura, K., and Saenger, W. (1998) Biochemistry37(11), 3654-64, Waldow, A., Schmidt, B., Dierks, T., von Bulow, R., andvon Figura, K. (1999) J Biol Chem 274(18), 12284-8).

Upon inspection of the 2-O sulfatase structure, several amino acids thatpotentially constitute the active site were identified (Table 3). Thereare several structurally conserved basic amino acids in the proximity ofFGly 82 including Arg 86, Lys 134, His 136 and Lys 308. The topology ofthe active site as observed in our structural model indicated that thecritical FGly 82 and the basic amino acid cluster are located at thebottom of a deep pocket (FIG. 10, Panel (B)). Such restrictive access tothe active site would appear to impose a clear structural constraint onthe substrate as it relates to the position of the 2-O sulfate groupwithin the oligosaccharide chain (i.e., externally vs. internallypositioned) upon which the enzyme acts. We predicted from this topologythat a sulfate group present at the non-reducing end of theoligosaccharide will be favorably positioned for catalysis; thejuxtaposition of an internal sulfate into the active site would requirea substantial bending of the oligosaccharide chain. Such chaindistortion would be sterically unfavorable. Based on these constraints,therefore, we predicted the sulfatase to hydrolyze 2-O sulfates in anexclusively exolytic fashion. This exclusivity for the non-reducing enddoes not necessarily preclude, however, the enzyme acting on longerchain oligosaccharides (i.e., those exceeding a disaccharide in length)provided that they in fact possess sulfates at the terminal 2-OHposition. The model does suggest a likely kinetic preference fordisaccharide substrates as they would most readily diffuse into and outof this narrow active site (see enzyme-substrate structural modelingbelow).

TABLE 3 Structure-based comparison of sulfatase active site residues 2-Osulfatase Arylsulfatase A Arylsulfatase B Arylsulfatase F. hepartaumHuman Human P. aeruginosa Cys-82 Cys-69 Cys-91 Cys-51 Arg-86 Arg-73Arg-95 Arg-55 Lys-134 Lys-123 Lys-145 Lys-113 His-136 His-125 His-147His-115 Lys-308 Lys-302 Lys-318 Lys-375 Gln-237 His-229 His-242 His-211Asp-42 Asp-29 Asp-53 Asp-13 Gln-43 Asp-30 Asp-54 Asp-14 Asp-295 Asp-281Asp-300 Asp-317 His-296 Asn-282 Asn-301 Asn318 Lys-238* Tyr-230 Glu-243Trp-212 Lys-175* Gln-153 Arg-180 Pro-161 Asp-159* His-151 Ser-172Ala-139 Thr-104* Val-91 Ile-113 — Glu-106 Val-93 Trp-115 — Lys-107*Pro-94 Pro-116 — Gln-309* Gly-303 Trp-319 Ala-376 Highly conserved aminoacids are listed in black. Non-conserved amino acids are listed in gray.Amino acids in the 2-O sulfatase that could be potentially involved insubstrate binding are noted by an asterisk. Structural alignment of themodeled 2-O sulfatase structure with the other sulfatases was obtainedbased on superposition of their Cα traces using the combinatorialextension algorithm (McLean, M. W., Bruce, J. S., Long, W. F., andWilliamson, F. B. (1984) Eur J Biochem 145(3), 607-15). Regions ofdeletion in the structural alignment are noted with a minus sign.

The surface of the active site pocket is comprised of many amino acidsthat can potentially interact with a disaccharide substrate. Theseinclude Lys 107, Lys 175, Lys 238, Gln 237 and Gln 309, Thr 104, Glu 106and Asp 159. Lysines and glutamines are commonly occurring amino acidsin heparin binding sites that interact with the sulfate and carboxylategroup. Unlike the amino acids proximal to the FGly 82, these residuesare not conserved in the other sulfatases that we examined (Table 3,denoted in gray), suggesting a potentially unique role of these aminoacids in dictating oligosaccharide substrate specificity. This disparityis particularly true when directly comparing the 2-O sulfatase andarylsulfatase A; many of the non-conserved amino acids in the 2-Osulfatase are charged while those in arylsulfatase A are predominantlyhydrophobic. This observation is consistent with the structuraldistinction of their respective substrates, i.e., the highly sulfatedHSGAG substrates of the 2-O sulfatase vs. the long hydrophobic alkylchains of cerebroside-3-sulfate substrate of arylsulfatase A.

Enzyme-substrate structural complex: Interaction between 2-O sulfataseand disaccharides—Since the active site can readily accommodatedisaccharide substrates, we modeled several unsaturatedglycosaminoglycan disaccharides. Our choice of Δ 4,5 unsaturatedsubstrates was logical for two reasons: 1) β-eliminative cleavage of aHS polysaccharide by the flavobacterial lyases that naturally occurs invivo results in the formation of disaccharides and other smalloligosaccharides all possessing a Δ4-5 unsaturated bond at thenon-reducing end uronic acid and; 2) the obligatory substrate-productrelationship between the 2-O sulfatase and the Δ 4,5 glycuronidase thatexists both in vitro and in vivo. A representative structural complexinvolving the trisulfated disaccharide ΔU_(2S)H_(NS,6S) (FIG. 11) wasused to describe the molecular interactions between the enzyme and thesubstrate. This choice was ultimately validated by the substratekinetics. A description of these interactions and their proposedfunctional roles is shown in Table 4. The functional roles of theconserved active site amino acids (listed in bold) were proposed basedon their interactions with the 2-O sulfate group and/or the geminal diolof the formylglycine at position 82. Identical roles have been proposedfor the corresponding amino acids in the three known sulfatase crystalstructures (Table 3).

TABLE 4 Functional assignment of 2-O-sulfatase active site amino acids-Active site Amino acids Proposed functional role Cys-82 Modified intohydrated form of the FGly - Oγ1 positioned for nucleophilic attack onsulfate group. Arg-86, His-136 Stabilizing the hydrated FGly byinteraction with Oγ2. His-136 is also positioned favorably forabstraction of proton from Oγ2 after catalysis to eliminate the sulfategroup and regenerate geminal diol. Lys-134, Lys-308, Gln- Coordinatewith the oxygen atoms of 2-O sulfate group to enhance electron density237 withdrawal from sulfate group thereby increasing theelectrophilicity of sulfur center. Lys308 is also positioned toprotonate the oxygen atom on the leaving substrate. Asp-295 Enhancesnucleophilicity of Oγ1 by proton donation. Lys-238, Lys-175 Interactionwith planar carboxyl group of ΔU may be critical for substraterecognition and positioning the 2-O sulfate group. Thr-104, Lys-107Interaction with 6-O sulfate on glucosamine may be critical forpositioning of 2-O sulfate group. Leu-390, Leu-391, Leu- Betterpositioned to make favorable hydrophobic contacts with the N-acetylgroup. 392 The amino acids listed in the first column were identified byinspection of the structural model presented in FIG. 3. The criticalactive site Cys-82 is indicated in boldface.

A closer inspection of the modeled enzyme-substrate complex revealedsome interesting possibilities pertaining to the role of thenon-conserved amino acids in substrate recognition and binding. Theplanar carboxylate group attached to the C5 atom of the Δ4-5 uronic acidis oriented in such a manner as to potentially interact with Lys 175,Lys 238. These amino acids could play an important role, therefore, infavorably orienting the 2-O sulfate within the active site. We werefurther interested in this arrangement given the additional constraintimposed upon the planar carboxyl group of the uronic acid by thepresence of the C4-C5 double bond. This constraint may further influencesubstrate orientation within the active site. Given this possibility, wepredicted a substrate discrimination exhibited by the 2-O sulfatasewhich is based on the presence of the Δ 4,5 double bond at theoligosaccharide non-reducing terminus. In the absence of this doublebond, the favorable orientation of the 2-O sulfate and the C5carboxylate afforded by charge interactions with Lys 178 and Lys 238,respectively, would not occur.

To better understand this likely structural constraint, we superimposedonto our trisulfated model substrate disaccharides containing anon-reducing end iduronic acid in either the ¹C₄ or ²S₀ conformation.The superimposition was such that the S—O—C2-C1 atoms of all the uronicacids coincided, thereby fixing the orientation of the 2-O sulfategroup. In this model, the carboxylate groups of the iduronic acidcontaining disaccharide substrates were, in fact, pointing away from theactive site pocket and were not positioned to interact as favorably withthe active site amino acids (i.e., Lys 175, Lys 238) as compared withthe original disaccharide substrate possessing a planar C5 carboxylate.

Our structural model of a sulfatase-trisulfated disaccharide complexalso points out key interactions involving additional sulfates (otherthan the uronic acid 2-OH position) present on the adjoiningglucosamine. In particular, the 6-O sulfate group interacts with thebasic side chain of Lys 107 within the enzyme active site (FIG. 11).This putative charge interaction would likely play an important role instabilizing the orientation of the substrate in the active site. Incontrast, the N-sulfate group of the disaccharide glucosamine isproximal to a contiguous stretch of leucines (390-392). In such anarrangement, it is the methyl group of an N-acetylated glucosaminerather than a sulfate at this position which is more likely to makefavorable hydrophobic contacts with these residues. This prediction wasborne out in one of our models docking the ΔU_(2S)H_(NAc,6S) substratein the active site.

We also modeled enzyme-substrate complexes containing two unsaturatedchondroitin sulfate disaccharides (ΔU_(2S)Gal_(NAc,4S) andΔU_(2S)Gal_(NAc,6S)). In comparison to our original model using theheparin disaccharide substrate, we found interactions with the 2-Osulfate and carboxyl group of the ΔU monosaccharide that were identicalto that of ΔU_(2S)H_(NS,6S). There were few interactions involving the4-sulfate and 6-sulfate groups, however. This particular model,therefore, does not exclude the ability of the so-called“heparin/heparan sulfate” 2-O sulfatase to hydrolyze 2-O sulfatedchondroitin disaccharides. Given a lack of additional favorable contactsbetween the enzyme and substrate (e.g., with either the 4-O or 6-Osulfates), we would anticipate a lower catalytic efficiency for thechondroitin disaccharides relative to the structurally correspondingheparin disaccharides.

In discussing this model, we must briefly consider the potential role ofdivalent metal ions. We decided not to include any such metal ions inour model of the 2-O sulfatase as we could find no divalent metalrequirement for enzymatic activity. A divalent metal ion is present,however, in all three sulfatase crystal structures that we examined. Ineach case, the metal-ion coordinates with the oxygen atoms of thesulfate group of the respective substrate. Additionally, a cluster offour highly conserved acidic amino acids has been observed to coordinatewith this divalent metal ion. In the case of human arylsulfatase B, forexample, the oxygen atoms of Asp 53, Asp 54, Asp 300 and Asn 301 arecoordinated with a Ca²⁺ ion. Three of the four-corresponding amino acidsin the flavobacterial sulfatase model that we have identified aspotentially coordinating with a metal ion are Asp 42, Gln 43 and Asp 295(Table 3). The fourth amino acid in the 2-O sulfatase correspondingspatially to Asn 301 of arylsulfatase B is His 296. The positive chargeof this position, however, does not favor the proximal location of adivalent metal cation. It is perhaps this unfavorable charge interactionwhich interferes with proper metal ion coordination.

Enzyme-substrate model: Mechanism for catalysis—Nearly identicalmechanisms for the hydrolysis of the sulfate ester bond involving theconserved active site amino acids have been proposed for humanarylsulfatases A and B and the bacterial sulfatase from Pseudomonasaeruginosa. The resting state of the active sulfatase in each of thecrystal structures is proposed to contain the geminal diol which isstabilized by interactions with basic residues. His 136 and Arg 86 ofthe flavobacterial enzyme are positioned appropriately in the activesite to do so (FIG. 10, Panel (B)). A critical step in catalysisinvolves the correct positioning the 2-O sulfate group such that thesulfur atom is accessible to the Oγ1 of the geminal diol. We havealready described how interactions of specific active site amino acidswith the planar carboxyl group of the uronic acid (Lys 175, Lys 238),with the 6-O sulfate of the glucosamine (e.g., Lys 107 and possibly Thr104) and with the 2-O sulfate itself (Lys 134, Lys 308) are likely toserve in this capacity (Table 4). At the same time, interaction of the2-O sulfate group with charged amino acids would also enhance anyelectron density withdrawal from the oxygen atoms, thereby increasingthe electrophilicity of the sulfur center. It has also been suggestedthat the nucleophilicity of the Oγ1 atom is enhanced by a possibleproton donation to a neighboring aspartic acid residue. In ourstructural model of the 2-O sulfatase, this residue would correspond toAsp 295.

An S_(N)2 mechanism may follow the above steps and eventually lead tothe cleavage of the sulfate ester bond. In this mechanism, the exocyclicoxygen atom on the leaving substrate may be protonated by water orpotentially by neighboring amino acids. In the 2-O sulfatase active sitemodel, Lys 308 is juxtaposed to protonate the leaving group (FIG. 11).The resulting sulfate group on the geminal diol is subsequentlyeliminated by abstraction of a proton from Oγ2 regenerating theformylglycine. His 136 is positioned to abstract this proton.

As we have already pointed out, our homology-based model of the 2-Osulfatase has several structure-function implications relating tosubstrate specificity. Many of these points are summarized in Table 4.When examined from the perspective of oligosaccharide structure, ourmodel addresses the issue of substrate specificity principally as itrelates to the following parameters: 1) the exolytic action of theenzyme; 2) the influence of oligosaccharide chain length; 3) thepresumed requirement for an unsaturated double bond at the non-reducingend; 4) the number and position of additional sulfates present withinthe glucosamine adjoining the 2-O sulfated uronic acid and; 5) thenature of the glycosidic linkage position between these twomonosaccharides. In the example which follows, each of these predictionsis empirically examined through biochemical and kinetic studies definingsubstrate preference.

Exolytic Action of the 2-O sulfatase—We addressed this importantquestion using as a substrate the purified heparin-derived AT-10decasaccharideΔU_(2S)H_(NS,6S)I_(2S)H_(NS,6S)I_(2S)H_(NS,6S)I_(HNAc,6S)GH_(NS,3S,6S).This oligosaccharide possesses a Δ 4,5 unsaturated uronic acid at thenon-reducing end and both externally and internally positioned 2-Osulfates. The substrate was first exhaustively treated with the 2-Osulfatase. The 2-O desulfated decasaccharide was then subjected to anexhaustive heparinase treatment. CE-based compositional analysesindicated the disappearance of the disaccharide ΔU_(2S)H_(NS,6S) by onlyone-third; two-thirds of this trisulfated disaccharide remained aftersequential treatment with the 2-O sulfatase and heparin lyases (FIG.12). Loss of a single sulfate was independently determined by massspectrometry. The loss of the single sulfate to the terminal 2-OHposition is suggested given the fact that the internally positionediduronic acid 2-O sulfates are structurally identical and shouldtherefore possess the same potential for desulfation. Based on thisassumption, the 2-O sulfatase would appear to act in an exolyticfashion. Our model clearly predicts a strong preference for sulfatespositioned at the non-reducing end where these sulfates would not beconstrained by the narrow topology of the enzyme active site.The requirement for an unsaturated Δ 4,5 non-reducing terminus—In arelated experiment, we assessed the ability of the 2-O sulfatase tohydrolyze size-fractionated hexasaccharides derived from the nitrousacid treatment of heparin. Unlike enzymatic cleavage, thesechemically-derived heparin saccharides do not possess a Δ 4,5unsaturated bond at their respective non-reducing ends. A majority ofthe resultant tetrasaccharides, however, do contain an I_(2S) at thisend. Using MALDI-MS, we were unable to detect any enzyme-dependentdesulfation of treated hexasaccharides. This result strongly suggests astructural requirement for the Δ 4,5 bond. The rationale for this isdescribed above in relation to our molecular modeling. In particular,the physical connection between this bond and the planar C5 carboxylateof the uronic acid carboxylate and how such a constraint permitscritical enzyme-substrate interactions for the proper orientation of the2-O sulfate within the enzyme active site was described.Determination of disaccharide substrate kinetics and specificity—We wereinterested in ascertaining any kinetic discrimination the enzyme maypossess for its disaccharide substrates based on the followingstructural considerations. 1) the number and position of sulfates on theadjoining hexosamine; 2) the glycosidic linkage position (i.e., β1→4versus α1→3); and 3) glucosamine vs. galactosamine as the adjoininghexosamine. We examined substrate saturation kinetics measured underMichaelis-Menten conditions. For these experiments, several heparindisaccharide substrates were used, each with a uronic acid possessing a2-O sulfate and a Δ 4,5 unsaturated bond at the non-reducing end, butdiffering in the degree of sulfation within the glucosamine. In additionthe two unsaturated chondroitin disaccharides ΔU_(2S)Gal_(NAc,4S) andΔU_(2S)Gal_(NAc,6S) were also examined as possible substrates. Theselatter two disaccharides differ from those derived from heparin/heparansulfate in possessing a β1→3 glycosidic linkage and a galactosamine inplace of a glucosamine. The results are summarized in FIG. 13 and Table5. All of the heparin disaccharides examined were hydrolyzed atsubstantial rates that included k_(cat) values which varied fromapproximately 600 to 1700 sec⁻¹. At the same time, the 2-O sulfatase didexhibit a substrate discrimination apparently based on the extent ofsulfation and largely manifested as a K_(m) effect. In particular, thepresence of a 6-O sulfate on the adjoining glucosamine conferred asignificantly lower K_(m) relative to its counterpart lacking such asulfate ester. In terms of catalytic efficiency, the trisulfateddisaccharide (ΔU_(2S)H_(NS,6S)) was clearly the preferred substratewhereas the mono-sulfated disaccharide (ΔU_(2S)H_(NAc)) was leastpreferred.

TABLE 5 2-O-sulfatase disaccharide substrate specificity Disaccharidek_(cat) (sec⁻¹) K_(m) (mM) k_(cat)/K_(m) ΔU_(2S)H_(Nac,6S) 1672 0.5153247 ΔU_(2S)H_(NS,6S) 814 0.087 9356 ΔU_(2S)H_(NS) 911 1.06 859ΔU_(2S)H_(Nac) 673 4.66 144 ΔU_(2S)Gal_(Nac,6S)* <100 >10 N.D. Kineticparameters were derived from a non-linear regressional analyses ofsubstrate saturation data depicted in FIG. 13. *Kinetic values for theunsaturated chondroitin disaccharide were approximated from doublereciprocal plots. N.D. not determined.

The 2-O sulfated chondroitin disaccharide ΔU_(2S)Gal_(NAc,6S) was onlyneglibly hydrolyzed under the same kinetic conditions. The enzyme diddesulfate this disaccharide to an appreciable extent, however, underreaction conditions involving a 4× higher enzyme concentration and alonger incubation time. Under these conditions, approximately 40% of thesubstrate was desulfated over a 20 minute period. In contrast, less than10% of chondroitin disaccharide ΔU_(2S)Gal_(NAc,4S) was hydrolyzedduring the same time period. To determine whether either or both ofthese 2-O sulfated chondroitin disaccharides could be quantitativelydesulfated under exhaustive conditions, we carried out an 18 hourincubation at 30° C. that included 5 mM of substrate and 5 μM enzyme.Under these conditions, both chondroitin disaccharides were greater than95% desulfated at the 2-O position. This result indicates that whilelinkage position and/or hexosamine isomerization are discriminatingkinetic factors, these physical parameters are not absolute determinantsfor 2-O sulfatase substrate recognition. It is interesting to considerthis latter observation in the context of the lysosomal pathway forglycosaminoglycan degradation in mammals where one enzyme desulfatesboth chondroitin and HS oligosaccharides at this position.

The apparent kinetic discrimination described above points to anunderlying structural determinant, namely a preference for glucosaminesulfated at the 6-OH and 2N positions. Our model does predict afavorable interaction with the 6-O sulfate in correct optimalorientation. At the same time, we would predict a bias in favor ofacetylation of the N-position rather than sulfation due to potentialhydrophobic interactions.

2-O sulfatase peptide mapping and chemical modification of active siteformylglycine—Finally, in describing the structure-function relationshipof the 2-O sulfatase active site, we come to the central catalyticplayer itself-the formylglycine at position 82. The recombinantexpression of catalytically active 2-O sulfatase in E. coli functionallyargues for this covalent modification of the active site in vivo. Weestablished the catalytic function of Cys 82 by site-directedmutagenesis. The mutant (C82A) was recombinantly expressed and purifiedas a histidine-tagged protein in the same manner employed for thewild-type enzyme. Comparable expression levels of soluble protein wereachieved. The C82A mutant, however, was completely inactive. Both thewild-type and mutant possessed the same secondary structure as exhibitedby their virtually superimposible CD spectra (FIG. 14), arguing againstany adverse global conformational changes induced by the molecularreplacement of the cysteine by alanine.

We also set out to demonstrate the physical presence of the FGly atposition 82 by the tandem use of protein chemistry and massspectrometry. 10 nanomoles of wild-type sulfatase (2-O ΔN¹⁻²⁴) and theC82A mutant were reacted with Texas red hydrazide (620.74 Da) asdescribed in Materials and Methods. The two sulfatase fractions weresubsequently trypsinized under mildly denaturing conditions followed byreductive methylation of the unmodified cysteines. The molecular massesof the resultant peptides were determined by MALDI-MS (FIG. 15). In thisexperiment, we identified a single ionized species uniquely present inthe labeled sulfatase experiment (FIG. 15, Panel (B)), but absent in theactive site mutant (FIG. 15, Panel (C)) or in the unlabeled control(FIG. 15, Panel (A)). The empirical mass of this species correspondedmost closely to the peptide sequence FTRAYCAQPLCTPSR (SEQ ID NO: 37)resultant from a partial trypsin cleavage. This peptide contains thesulfatase consensus sequence CXPXR which includes the critical activesite cysteine (denoted in bold) at position 82. The mass of this peptideis consistent with first the conversion of this cysteine to aformylglycine (FGly 82) followed by the covalent hydrazone linkage ofthe aldehyde-reactive fluorophore at this position. It also takes intoaccount the carbamidomethylation of the second (unmodified) cysteinepresent in this peptide. These data, taken together with the loss offunction observed for the C82A mutant, establish the importantstructure-function relationship for this active site modification.

Each of the foregoing patents, patent applications and references thatare recited in this application are herein incorporated in theirentirety by reference. Having described the presently preferredembodiments, and in accordance with the present invention, it isbelieved that other modifications, variations and changes will besuggested to those skilled in the art in view of the teachings set forthherein. It is, therefore, to be understood that all such variations,modifications, and changes are believed to fall within the scope of thepresent invention as defined by the appended claims.

1. An isolated nucleic acid molecule selected from the group consistingof: (a) nucleic acid molecules which hybridize under stringentconditions to a nucleic acid molecule having a nucleotide sequenceselected from the group consisting of nucleotide sequences set forth asSEQ ID NOs: 1 and 3, and which code for a 2-O sulfatase, wherein thehybridization conditions are 1) hybridization at 65° C. in hybridizationbuffer that consists of 3.5×SSC, 0.02% Ficoll, 0.02% polyvinylpyrrolidone, 0.02% Bovine Serum Albumin, 2.5 mM NaH2PO4, pH 7, 0.5%sodium dodecyl sulphate (SDS), 2 mM ethylenediaminetetracetic acid(EDTA), wherein SSC is 0.15M sodium chloride/0.015M sodium citrate, pH 7and 2) washing in 2×SSC at room temperature and then in 0.1×SSC/0.1×SDSat 68° C., (b) nucleic acid molecules that differ from the nucleic acidmolecules of (a) in codon sequence due to degeneracy of the geneticcode, and (c) full complements of (a) or (b).
 2. The isolated nucleicacid molecule of claim 1, wherein the isolated nucleic acid moleculecomprises a nucleic acid sequence set forth as SEQ ID NO:
 3. 3. Theisolated nucleic acid molecule of claim 1, wherein the isolated nucleicacid molecule codes for SEQ ID NO:
 4. 4. An isolated nucleic acidmolecule comprising a nucleotide sequence that is at least 90% identicalto a nucleotide sequence selected from the group consisting of SEQ IDNOs: 1 and 3, which codes for a 2-O sulfatase that has an activity ofthe 2-O sulfatase set forth as SEQ ID NO: 2 or
 4. 5. The isolatednucleic acid molecule of claim 4, wherein the nucleotide sequence is atleast 95% identical.
 6. The isolated nucleic acid molecule of claim 5,wherein the nucleotide sequence is at least 97% identical.
 7. Theisolated nucleic acid molecule of claim 6, wherein the nucleotidesequence is at least 98% identical.
 8. The isolated nucleic acidmolecule of claim 7, wherein the nucleotide sequence is at least 99%identical.
 9. The isolated nucleic acid molecule of claim 8, wherein thenucleotide sequence is at least 99.5% identical.
 10. The isolatednucleic acid molecule of claim 9, wherein the nucleotide sequence is atleast 99.9% identical.
 11. An expression vector comprising the isolatednucleic acid molecule of claim 1 operably linked to a promoter.
 12. Anexpression vector comprising the isolated nucleic acid molecule of claim4 operably linked to a promoter.
 13. A host cell comprising theexpression vector of claim
 11. 14. A host cell comprising the expressionvector of claim
 12. 15. A composition comprising the nucleic acid ofclaim 1 and a pharmaceutically acceptable carrier.
 16. A compositioncomprising the nucleic acid of claim 4 and a pharmaceutically acceptablecarrier.
 17. A composition comprising: the expression vector of claim 11and a pharmaceutically acceptable carrier.
 18. A composition comprising:the expression vector of claim 12 and a pharmaceutically acceptablecarrier.
 19. A composition comprising: the host cell of claim 13 and apharmaceutically acceptable carrier.
 20. A composition comprising: thehost cell of claim 14 and a pharmaceutically acceptable carrier.